Protein Chromatography: A Concise Guide to Ion Exchange Chromatography

Protein Chromatography
Protein properties are directly affected by their environment, which depends on pH, temperature,
solvent effects, and pressure. With these affects has come more desire for better techniques in the
purification and separation processes. These processes hopefully will prevent loss of biological
activity and denaturation. With ion exchange chromatography comes many advantages over column
chromatography such as union between the target protein and functional groups at a very quick rate,
exponential growth without difficulty, short processing times, no heating, no extreme pH range, and
a multitude of other things (Santos et al. 2012). Ion exchange chromatography in short is ideal due
to the low cost, longevity through harsh cleanings, and high ... Show more content on
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The first included Cibacron Blue Sepharose CL–6B, proteins, and egg white lysozyme. They tested
the proteins and egg whites binding ability to the Cibacron Blue. To make matters easier, they only
searched for absorption of pure proteins from 0.05 M Tris–HCl buffer, pH 7.2. This was achieved by
eluting the absorbed proteins with 1M KCl In in 0.05 M Tris–HCl buffer. The second type included
immobilized monoclonal antibodies and the absorption of E. Coli B–galactosidase against B–
galactosidase. This was chosen due to the simplicity of measuring the activity of B–galactosidase
that could then tell us the concentration of the antigen. The only downside to this approach was B–
galactosidase has a much higher molecular weight that most antigens commonly used by this
method. This means the large molecule will have trouble penetrating the pores in the immuno–
absorbents. This was done by immobilizing the antibodies on a silica–based adsorbent, which was
then added to PBS buffer. The buffer contained antibodies. The flask they were in was then shaken
for four hours, followed by removal of all the unreacted protein that was done by using a funnel
with sodium chloride solution. To wash the gel they used propionic acid and then stored the
adsorbent in PBS buffer. Finally to elute the absorbed B–galactosidase, they used 6 M urea in 20
mM Tris–HCl, 10 mM ethylene diamine tetraacetic acid, and 10 mM sodium Chloride, 0.1 M 2–
mercaptoethanol, pH 7.2 (Chase
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Measuring A Computational Prediction Method For Fast And...
In general, the gap is broadening rapidly between the number of known protein sequences and the
number of known protein structural classes. To overcome this crisis, it is essential to develop a
computational prediction method for fast and precisely determining the protein structural class.
Based on the predicted secondary structure information, the protein structural classes are predicted.
To evaluate the performance of the proposed algorithm with the existing algorithms, four datasets,
namely 25PDB, 1189, D640 and FC699 are used. In this work, an Improved Support Vector
Machine (ISVM) is proposed to predict the protein structural classes. The comparison of results
indicates that Improved Support Vector Machine (ISVM) predicts more accurate protein structural
class than the existing algorithms.
Keywords–Protein structural class, Support Vector Machine (SVM), Naïve Bayes, Improved
Support Vector Machine (ISVM), 25PDB, 1189, D640 and FC699.
I. INTRODUCTION (HEADING 1)
Usually, the proteins are classified into one of the four structural classes such as, all–α, all–β, α+β,
α/β. So far, several algorithms and efforts have been made to deal with this problem. There are two
steps involved in predicting protein structural classes. They are, i) Protein feature representation and
ii) Design of algorithm for classification. In earlier studies, the protein sequence features can be
represented in different ways such as, Functional Domain Composition (Chou And Cai, 2004),
Amino Acids

The Importance Of Green Fluorescent Protein
In the 1960's, Green Fluorescent Protein was discovered to be responsible for Aequorea victoria's
fluorescence under UV light (Niwa et al., 1996). Today, GFP is often used in protein tagging and has
made it possible for scientists to study expression and track proteins in vivo. With the rising
importance of GFP fusion proteins and other recombinant vectors, the metal affinity of Histidine
helps to make the protein purification process easier (Lilius et al., 1991). His tags are commonly
used to purify proteins through immobilized metal–affinity chromatography (IMAC) (Lilius et al.,
1991). This rapid and efficient method separates the recombinant protein from unwanted products
such as RNA. The His tag DNA sequence is inserted into the ... Show more content on
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It is of strong interest to molecular biologists because of the strong light emission of 508 nm under
UV light. UV wavelengths excite the fluorophore, contained in the Ser – Tyr – Gly sequence near
GFP's N–terminus, and a bright green fluorescence is given off as a result (Niwa et al., 1996). GFP
can be successfully tagged to many proteins without disturbing their function (Feilmeier et al.,
2000). This allows scientists to exploit the protein for its usefulness in marking proteins in vivo.
GFP tagging has many applications. It can be used to study gene expression by inserting gfp after a
promotor in the plasmid, so that if expressed, the colony expressing the gene of interest can be
identified by a green fluorescence of GFP under UV light (Feilmeier et al., 2000). Specific cellular
organelles can be tagged, such as the endoplasmic reticulum, to visualize protein localization and
networking inside the living cell. GFP can also be used to monitor cofactor levels within a cell in
real time. For example, when GFP was tagged to proteins that contain a receptor sensitive to Ca2+,
they interacted in a way so that the fluorescence of the fusion protein was proportional to the
calcium ion concentration with less than a 1 second lag in the cell (Romoser et al., 1997).
Throughout the decades, GFP mutants have been created to give brighter emission and different
wavelengths. Researchers can track separate cell pathways at the same time, since different proteins
can

Gel Electrophoresis Lab Report
Introduction:
This experiment will be done as the first step in determining an unknown protein. The second part of
the protein determination will be done using the Western blot technique. We will use the Sodium
Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE) method to separate proteins by
their molecular weight. This method is often used to determine the purity and weight of a protein.
Electrophoresis is a method of separating proteins based on their chemical and physical properties.
Gel electrophoresis has a gel support mediums that includes polyacrylamide, starch and agarose.
The gel is chemically inert, it is easy to handle, and can me made to fit a desirable porosity. This
makes it good to use for molecules with high molecular weights. After separating the gel, it will be
stained with Coomassie Blue to visualize the protein. ... Show more content on Helpwriting.net ...
Heat linearizes the protein, and the reducing agents break the disulfide bonds that maintain the
protein structure in native form. To ensure that the proteins will be separated by their molecular
weights and not their charge, SDS will be used to coat the protein and give it a negative charge. This
ensures that the protein is separated just by their molecular weights. It is important to note that if
catalytic protein (enzymes) are being separated, they should not be treated with reducing agents or
SDS. Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis or SDS–PAGE, forms a protein
complex by binding to the polypeptides in the proteins. This SDS–protein complex is proportional
to the proteins molecular weight. Biochemist can use this information to identify key proteins in a
biochemical system. Dr. Sudakshina Ghosh uses this technique, for example, to "identify specific
oxidized proteins that provide mechanistic information regarding oxidative stress" (1

Bovine Blood Lab
For this experiment, whole bovine blood was used. The first process was to separate the blood into
cellular and plasma fractions. 100 µL of whole bovine blood was transferred into a yellow
microcentrifuge tube that was labeled WB using a P–200. 50 µL of whole blood was added to a blue
microcentrifuge tube labeled WB. Both tubes were capped and placed in ice. 2 mL of the remaining
blood was transferred into a Clear 2 mL tube using a P–2000 and centrifuged for 5 minutes at 8000
RPM. Afterward, 800 µL of the supernatant from the Clear tube was transferred into a yellow tube
labeled WP and 50 µL of the supernatant was added to a blue tube labeled WP. These tubes were
then capped and kept on ice.
Next, the plasma proteins from the blood were isolated ... Show more content on Helpwriting.net ...
A BSA standard serial dilution was created in wells A1–F1. Fifty microliters of dH20 were added to
each well and fifty microliters of the BSA stock was added to A1 and mixed. Fifty microliters from
A1 was transferred to B1 and this was repeated for C1, D1, and E1. In wells A2–A4, triplicates were
created by transferring 10 microliters from A1. Triplicates were created for the other BSA standards
using the same amount, but with a different tip for each standard. The samples WP, WB, Es, and Ap
(yellow tubes) were also used in the Bradford Assay. The dilutions for the samples were 1:10, 1:100,
1:500, and 1:1000. The first dilution consisted of 90 microliters of water and 10 microliters of the
sample. The second dilution consisted of 90 microliters of water and 10 microliters of the sample as
well. The third dilution composed of 80 microliters of water and 20 microliters of the sample. The
last dilution composed of 50 microliters of water and 50 microliters of the sample. These dilutions
were used for each sample and triplicates were generated for each dilution. Two hundred microliters
were added to each triplicated well for all the samples, including the standard. Then, the microplate
was placed on the spectrophotometer and the absorbance was

Summary Of Mut-OX In A549 Cells
In this SA, we will study translocation of DNA damage repair proteins from the cytoplasm to
mitochondria in response to mtDNA damage induced by mt–OX in A549 cells. We will treat A549
cells with mt–OX as mentioned in SA#...., followed by using a SILAC method as described above.
We will perform differential ultracentrifugation to isolate cellular fractions as previously described
(18). The purity of cytoplasmic and mitochondrial fractions will be assessed by western blotting
using IκB–α and Tom20 markers, respectively. We expect that mtDNA damage induced by mt–OX
will cause translocation of DNA repair proteins from the cytoplasm to mitochondria. Moreover, it
was reported that more than 30 posttranslational modifications including the most ... Show more
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to determine viability, amount of mitochondria, and mtDNA damage by RT–PCR. Relative levels of
mtDNA damage will be evaluated by a quantitative method using amplification of mtDNA
fragments of different lengths. Results will be presented as a ratio of long versus short fragments as
shown in Fig. 10 (13, 14).
Studies in human primary ATII cells: We will assess the function of identified translocated
mitochondrial proteins in human ATII cells. We will validate obtained results using these unique
cells isolated from control lung donors and patients with emphysema. We found in our preliminary
data that ATII cells isolated from individuals with this disease have more mtDNA damage and less
mitochondria amount than controls (Fig. 10). We will check expression of identified proteins and
genes by western blotting and RT–PCR, respectively in cytoplasmic and mitochondrial fractions. We
expect to identify dysregulated proteins which are involved in mtDNA damage repair in primary
ATII cells isolated from patients with emphysema.
We will also knockdown identified genes in control ATII cells using Sendai virus strategy as
previously reported (21) followed by treatment with mt–OX as described in SA#1.1. We will also
overexpress selected genes using adenovirus construct as we previously reported (7) to complement
this study. Moreover, we will analyze mtDNA damage and mitochondria amount as mentioned in
SA#..... We expect to detect high sensitivity of mtDNA to damage induced

The Protein Status Of Children With Protein Energy...
1– How would you evaluate the protein status of children with PEM? Which markers would you use
and what are the expected results?
The protein status of children with protein energy malnutrition (PEM) can be assessed by complete
patient history and physical examination in which the skin would appear thinner and dull/discolored,
hair growth is restarted and hair may fall and there would be loss of lean body mass over long
period of time (Stipanuk and Caudill). The WHO recommends laboratory tests of blood glucose,
microscopic blood smears, hemoglobin, urine and stool examination& culture for ova and parasites,
serum albumin, and measurement of electrolytes (Lin et al.). The most commonly used method to
assess PEM is measurement of plasma ... Show more content on Helpwriting.net ...
The measurement of antioxidant status can also evaluate the PEM. Khare et al. concluded that stress
is present in PEM as a result of free reactive oxygen radicles and these cause membrane lipid
peroxidation and thus change in oxidant and antioxidant levels in the body may predict the disease
grading in PEM (Khare et al.)
2– Why nitrogen balance studies are considered to be better than the factorial method to estimate
protein requirements?
Although both methods have some limitations but the nitrogen balance studies are considered to be
better than the factorial method to estimate protein requirements because the factorial approach has
some uncertainties and assumptions involved during measurement of protein requirement
(Allowances). In many long term studies the nitrogen balance method is considered as most reliable
and better evidence to determine the protein requirement (Rand, Pellett and Young). The nitrogen
balance studies have linear relationship between the nitrogen balance and nitrogen intake and this in
not linear in factorial method (Morales, Craig and MacLean). In nitrogen method the direct
measurements of nitrogen balance have been implicated to measure the protein requirement during
disease and heathy conditions (Pencharz). This is not feasible by the factorial method to directly

Materials And Methods Of Chemicals
Materials and methods
Chemicals used:
Fipronil (Insecto SC 5%) is a product of BASF Company and manufactured by Sinochem Group–
Ningbo Technical Co. Ltd, China. Zinc was obtained (in the form of zinc sulphate heptahydrate)
from October Pharma, Egypt. The kits used for the following biochemical assays were purchased
from Biodiagnostic Company, Dokki, Giza, Egypt: superoxide dismutase (SOD, EC 1.15.1.1),
catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GPx, EC 1.11.1.9), glutathione–s–transferase
(GST, EC 2.5.1.13), glutathione reduced (GSH), lipid peroxidation (LPO) and total protein. All
other chemicals were obtained from reputed companies.
Animals and experimental design
Twenty male albino rats, weighing 90 ±10 g were used in this study. The animals were obtained
from the animal house of the National Organization for Drug Control and Research (NODCAR),
Dokki, Giza, Egypt. They were housed under normal environmental conditions of temperature and
humidity and allowed to adapt to the new environment for two weeks before starting the experiment.
Animal rooms (23±2 Cº) were maintained on a 12:12h light/dark photoperiod. Animals were
provided with food with free access standard pellet diet and water ad libitum. All animal procedures
were conducted according to accepted standards of animal care following NODCAR Guidelines.
Rats were randomly divided into four groups, five rats in each group. Control group, rats received
drinking water. Zinc group, rats received zinc at

. E. V, And Blundell, T. L )
Pires, D. E. V., Ascher, D. B., & Blundell, T. L. (2014). DUET: a server for predicting effects of
mutations on protein stability using an integrated computational approach. Nucleic Acids
Research, 42(W1), W314–W319. https://doi.org/10.1093/nar/gku411
The problem that was addressed by Pires, Ascher, and Blundell was the generation of meticulous
computer simulations to analyze the effects of non–synonymous single–nucleotide polymorphisms
(nsSNPs) on the ever–expanding proteome (2014). It was hypothesized that if the system integrated
both the mCSM and SDM techniques, then employed a predictor run by Sequential Minimal
Optimization to analyze which outcome is most probable, then the final simulations should be
accurate more frequently ... Show more content on Helpwriting.net ...
The Pearson coefficient of correlation and the standard error values are present in the top left of both
graphs, symbolized as ρ and σ respectively. The coefficient of correlation for the training set was
0.74 illustrating a strong positive correlation between the actual variation in protein stability and that
which was predicted by DUET, with a standard error of 0.98 kilocalories per mole. The blind test
demonstrated strong but moderate correlation between these variables with a coefficient of
correlation of 0.68 and standard error only being 1.39 kCal/mol; likewise, the p53 protein mutations
test showed strong correlation with a coefficient of 0.71 and error standards of 1.13 kCal/mol. Such
results were later defined to be significantly more reliable than that of other simulators (Pires et al.,
2014).
Figure 2: This figure depicts the predictions created by DUET; including the variation in protein
stability, expressed in Gibbs Free Energy (ΔΔG) in kilocalories per mole, due to the presence of a
specific nsSNP (Pires et al., 2014). The difference in Gibbs Free Energy (ΔΔG) is represented three
times; once as calculated by the mCSM technique (i), once by the SDM method (ii), and the final
DUET calculation (iii) which combines the previous two (Pires et al., 2014). The values that are
displayed

P53 Protein Lab Report
1. A. Prediction: If elephants have multiple copies of functional p53 genes, then elephants will have
a lower rate for developing cancer when compared to organisms with less copies of p53 genes.
2. Could culture cells of both humans and elephants in medium and compare to see which showed
higher levels of expression of the p53 gene.
First the p53 protein would need to be isolated. The simplest method for identifying proteins that
bind to one another tightly is co–immunoprecipitation. In this technique, an antibody is used to
capture and precipitate a specific target protein from an extract prepared by breaking open cells. If
p53 is associated tightly with another protein, the partner protein will precipitate as well. Use
antibodies against p53 ... Show more content on Helpwriting.net ...
We could use induced pluripotent stem cells, where adult cells are taken into an ES–like state by
artificially driving the expression of a set of three transcription regulators called Oct3/4, Sox2, and
Klf4. This converts fibroblasts into cells with practically all the properties of ES cells, including the
ability to proliferate indefinitely and differentiate in varied ways to contribute to any tissue.
Problems are that the conversion is lower and only a small proportion of fibroblasts make the switch
and it may not be safe with such an abnormal developmental history to transfuse these cells back
into

Protein Adsorption Using Fluorescently Tagged Rh Fgn
Introduction:
For this laboratory, we are studying protein adsorption using fluorescently tagged Rh–FGN. To
prepare for this study, model sutures of a specific length have been coated in albumin, which allows
us to compare the difference between a coated suture and an uncoated suture in regards to "sticky"
protein adsorption. Based on the effects of surface energy (γ) on protein adsorption, the uncoated
nylon model suture will yield higher protein adsorption then coated suture, since the coated suture
should have higher surface energy. Thus at a 0.05 assurance level (α = 0.05) our null hypothesis is as
follows: there is not a difference in protein adsorption between uncoated and coated nylon model
sutures when exposed to a fibrinogen solution. Our alternative hypothesis (Ha) is as follows: there is
a difference in protein adsorption between uncoated and coated nylon model sutures when exposed
to fibrinogen solution at a 0.05 assurance level (α = 0.05).
Background:
When designing new state–of–the–art biomedical devices, the reaction between the biomaterials
placed in the body and the body itself is highly important and widely studied. The compatibility of
biomaterials governs their effectiveness; therefore, understanding the mechanisms of these
interactions and the thermodynamics behind them is essential for creating and improving these
devices. When determining the materials necessary for these devices, protein adsorption must be
addressed, due to the fact that

Role Of Proteins
Proteins play a role in almost every physiological process that takes place in our body and
understanding protein functionality can lead to a better understanding human biology and life itself.
The central dogma of biology describes the flow of genetic information from DNA to RNA to
proteins. It has long been established that the structure of the protein determines the function,
however this idea has been challenged in recent years by the discovery of intrinsically disordered
proteins. Protein structure determines the key characteristics exhibited by the protein and can
determine its role in biological processes. Proteins are comprised of amino acids; this sequence of
amino acid residues describes the protein's primary structure. The ... Show more content on
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Officially, intrinsically disordered proteins are proteins that do not have a fixed three–dimensional
conformation. Whether the entire protein is disordered or only certain domains within a protein are
disordered, they do not fold into a stable well–defined tertiary structure. Some proteins may be
ordered overall but contain a disordered sequence, which is commonly called an intrinsically
disordered region (IDR). This unique flexibility of structure allows for many advantages in
biological functions, some well known and others still being discovered. While research into IDPs
are ongoing, research shows that IDPs play huge roles in cell signaling and regulation. To fully
understand the function of intrinsically disordered proteins, it is best to first understand what
accounts for their unique structures.
There are numerous methods researchers utilize to determine if a protein is or has a disordered
region. The most prevalent is X–ray crystallography, which was the method used to first discover
disordered protein domains. X–ray crystallography allows for a visualization of a protein's three–
dimensional structure through the use of x–ray diffraction. When a protein is disordered, it possesses
a lower or nonexistent electron density that can be detected through this process. Even early on
when the first protein crystal structures were discovered, there were protein

Functions Of Eukaryotic Cells For Various Functions And...
Microtubules are an essential part of eukaryotic cells for various functions and processes, and are
located throughout the cytoplasm in the cytoskeleton. Microtubules are essential for multiple
cellular processes such as, cellular division and cellular movement and transport(6). Since
microtubules are required for many cellular processes, mutations involving microtubules can cause
various diseases including neurodegenerative diseases and cancer(5). One of the most common
neurodegenerative diseases involving mutations of microtubules is Alzheimer's Disease. Alzheimer's
causes the neuron cells in the brain lose function and eventually die causing the brain to
deteriorate(1). Microtubules affect this disease by their associated proteins, ... Show more content on
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One of the types of tubulin is acetylated tubulin, and are located on stable microtubules. The
stability of an acetylated protein allows the protein to become a marker for macromolecule and
organelle transportation in neurons(2). It was hypothesized that the zebrafish brain would have a
higher concentration of acetylated tubulin than in the zebrafish eye.
Methods
Protein Extraction Tubes containing zebrafish brain or eye tissue and 500μl of extraction buffer
were obtained and tissue was grinded to a slurry and homogenized. The homogenate was placed in a
100C hot block for 1 minute. The sample was vortexed for 5 minutes and spun at 14,000 RPM for a
total of 30 minutes. The 200μl of supernatant was removed and put into a tube and stored at –80C
for a week. The rest of the supernatant was also placed in a separate tube and labeled as S1. The
supernatant was resuspended in 500μl of the previously used extraction buffer and vortexed. A large
amount of the solution was removed and put into a tube labeled P1 and was also kept at –80C for 1
week.
Protein Quantification by the Bradford Assay The S1 and P1 samples underwent serial dilutions to
create a total of 3 tubes per sample. Fifteen cuvettes were gathered and 30μl of each sample was
added to the cuvettes. The first cuvette was filled with 1.5 mL of Coomaisse Reagent. Coomaise
reagent was added to each cuvette, with one minute intervals between each measurement to allow
the protein mixed with

Protein Binder For Affinity Purification Of Human...
Protein Binder for Affinity Purification of Human Immunoglobulin Antibodies
Background
Antibodies are used for many different purposes. According to
WORLDPHARMACEUTICALFRONTIERS, 2014 saw $75 billion dollars spent on monoclonal
antibodies which is more than half of the biopharmaceutical market 1. For this reason, novel
methods of protein purification are needed to address the growing demand for antibodies. In this
paper, the research group provides a novel method of purifying human immunoglobulin protein
(IgG) by the use of a "repebody".
According to their previous research, a "repebody" is nonantibody protein structure containing
consensus designed leucine rich repeat modules.2 This particular protein was recently discovered in
... Show more content on Helpwriting.net ...
Protein A is thermostable, and is not destroyed by trypsin4; however, Protein A is most useful for its
stability in regard to pH. It's polypeptide structure is so stable that it has continuous stability both at
very acidic pH (0.99) and at a very basic pH (11.8) 3,4. For this reason, it useful across many pH
levels that would be useful in a protein affinity assay because this particular assay requires changing
the pH to determine what would be the best to elute the protein that is being selected for; however,
for an affinity assay involving human immunoglobulin, protein A is used for an additional reason.
Protein A is from a bacteria and human immunoglobulin is associated with the immune system of
the humans, as the name implies. Protein A contains five locations that it can bind to IgG5. These
five domains, often labeled regions A–E, each consist of 58–62 amino acid residues, and a C–
terminal consists of 150 amino acid residues. The IgG binding domains (A–E) each consist of three
anti–parallel α–helices which are stabilized by hydrophobic regions between them. These domains
bind with the Fc region of the immunoglobulin and form a complex with the Fc region. When this
binding occurs a conformational change occurs which is reinforced by polar, and hydrophobic
interactions4. This binding pattern is the basis for its use in protein affinity assay that involve eluting
IgG out of solution that contain other human immunoglobulins. As the other

Materials And Methods Of Protein Sequences Of Lassa Virus
Materials and Methods
Retrieving protein sequences:
Available all the protein sequences of Lassa virus were retrieved in FASTA format from
UniProtKB(http://www.uniprot.org). Organisms other than Lassa virus and strains of Lassa virus
causing effect on organisms other than human were excluded.
Prediction of the most potent antigenic protein:
Every retrieved FASTA sequence of corresponding protein was submitted in VaxiJen v2.0 [33] in
plain format to identify the antigenic score of each protein. During this process, the default
parameters (THRESHOLD=0.4, Sequence Output) were used and virus was selected as target
organism. The protein with highest antigenic score was taken for further evaluation. Protein with
less than 120 amino acid sequences was not considered for this prediction.
Prediction of T cell epitope:
Cytotoxic (CD8+) T cell epitopes as MHC class I ligand were predicted using NetCTL 1.2 [34], a
server based on neural network architecture, for each of the 12 HLA class I super types defined by
Lund et al. in [35] (A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, B62). Previously selected
FASTA sequence of the polymerase protein was submitted to that server with the parameter of
threshold level set at 0.5 to have sensitivity and specificity of 0.89 and 0.94 respectively. This would
be helpful for finding more epitopes effectively. An integrated value of transporter of antigenic
peptide (TAP) transport efficiency, MHC–I binding and proteosomal cleavage efficiency

Purification Of Purification And Purification
When examining the purification data in Table 4, we can analyze the different methods of
purification of LDH and determine how effective each methods is in order to ultimately purify LDH.
Unfortunately, there are two different definitions of purification: one is have a high end yield of the
protein, while the other is having a high end concentration without any contaminating proteins.
Furthermore, in order to achieve one type of purification, the other one has to be given up. The very
first purification step involved the 65% ammonium sulfate cut that resulted in a highest protein
concentration out of all the purification steps. The protein concentration went up from 3.925mg/ml
from the clarified homogenate to 28.11mg/ml, which is resulted of the pelleting out of the LDH and
reducing the volume from 114mL to 5.7mL. Furthermore, the 65% pellet cut was able to recovery
46% of the LDH, while purifying it by 6.8 fold. Also, this step was able to remove 65% of the total
proteins, while retaining 35% of it. Due to the increased purity and 46% recovery of LDH we can
conclude that most of the removed total protein was non–LDH, contaminating protiens.
Unfortunately, since this was only the first purification step, the amount of comtaminating proteins
is still fairly high as depicted in Figure 14, where the 65% cut pellet contains several dark bands
besides the one at 35kDa. The second purification step involved the affinity chromatography column
that resulted in a sharp decrease

Advantages And Disadvantages Of Protein
Advantages:
Can be used to estimate multiple proteins in a mixture of proteins
If the given sample is a mixture of proteins, the proteins are also separated for further use, for
example the separated proteins can then be used for mass spectroscopic studies
Disadvantages:
Length process and needs to be done carefully
To obtain a valid comparison for the unknown sample response to that of the known standard, the
data must be acquired and processed under identical conditions.
3. Determination by weight
3.1. Determination by dry weight
A simple but lengthy method for the estimation of the protein, determination by dry weight gives a
very accurate amount of the protein in a given sample. Drying a protein–containing sample in a
104ºC to ... Show more content on Helpwriting.net ...
The mass spectrometer attains this by first ionising the sample, with the help of chemicals,
bombarding with other ions or by using a laser (MALDI), and then passing the ions through an
electric and magnetic field to deflect it towards a detector. The strengths of the electric and magnetic
field are varied to focus ions with a specific mass by charge ratio. The output, i.e. the mass
spectrum, is a plot of the ion signal vs the charge by mass ratio. This plot is characteristic to proteins
and can be used for the identification and

The Light Energy Absorbed By Colored Compounds
Spectrophotometry is the process by which the light energy absorbed by colored compounds, is
determined and used for quantitative and qualitative analyses. We use this process to often produce a
standard curve, and use that to compare absorbance with different factors. In Experiment 3.1–3.4,
spectrophotometry is used to produce the absorbance spectrums for the basic and acidic forms of
PNP, to determine the pKa of PNP, and to develop standard curves for determination of unknown
concentration and pH of unknown samples.
Quantification of proteins is needed to determine the progress of protein purification. As the protein
becomes more purified, its specific activity will increase as well. In Experiment 4.1, dilutions of
PNP are prepared, and the Bradford Method was used to measure protein concentration.
There are different types of chromatography, and we would be using gel filtration and affinity.
Chromatography is defined as the separation of molecules based on properties such as molecular
mass, charges or solubility. In Experiment 5.1, gel filtration chromatography was used to separate
molecules based on their molecular mass. The stationary phase in the column contains tiny pores,
those that are unable to fit into the pores are eluded first; thus, the small molecules are eluded last.
We are able to witness a visual demonstration of gel filtration chromatography, the separation of
three different colored compounds: blue dextran, cytochrome c, and potassium chromate. In

Rubisco Lab Report
Introduction Proteins are critical components in understanding cells and organisms which can
contribute to further developments in medicine. Proteins compose more than 50% of the dry weight
of cells [2]. Enzymes are the proteins that catalyze most reactions in a cell that keeps the cell going.
By isolating protein scientists can understand, modify, and sequence specific proteins away from
other cellular components. The specific protein being looked at is Rubisco (Ribulose–1,5–
bisphosphate carboxylase/ oxygenase) which is a plant enzyme that has a key role in photosynthesis
[1]. Rubisco is defined by its two polypeptide subunits at 55kDa and 14kDa. The aim of this
experiment is to successfully isolate a purified form of the protein Rubisco. In order to for this occur
a series of methods for isolation are done by separating the different proteins present from Rubisco
according to their solubility, charge, and size. Since Rubisco is very soluble when ammonium
sulfate reaches 50% saturation Rubisco should be present and can then be isolated when ion
exchange chromatography and protein electrophoresis is done due to the fact that Rubisco is
negatively charged ... Show more content on Helpwriting.net ...
The filter flask, vacuum, and SNAP i.d. 2.0 were securely connected with tubing. The proteins were
transferred to a membrane and was incubated with primary antibody. The primary antibody was then
washed three times. Afterwards a secondary antibody was added and the same procedure was
repeated. 10mL of 5–bromo–4–chloro–3–indoyl phosphate/ nitro blue tetrazolium chloride
chromogenic substrate solution was added to the blot which was then wrapped with aluminum foil
and labeled. It was transferred to the rotator table and rotated for periods of 15 minutes until bands
formed. Since no bands formed it was left to rotate overnight. The membrane was then paper
toweled dry and the bands were measured and used to create a standard

Tracing Chemical Evolution of the Human Body Essays
Introduction:
Evolutionary pathways can be traced through several different means. Two of the most popular are
looking at the phenotypic signs like body structure or appendages things that can be seen, another
popular way of tracing is similar structures that have developed or gone away like a tail bone on a
human. Another way to track evolution is to look at the development of proteins and other chemicals
in the organism's body.
Through tracking the evolution of chemicals a person can see how close two organisms are related.
This method of tracking is useful with proteins like Pax6 a gene that controls eye development.
Organisms that have an eyes can be found to have a variation of Pax6, these organisms include
mice, humans, flies, ... Show more content on Helpwriting.net ...
After the website is pulled up it will show basic information on arrestin afterwards preform a
BLAST search in the human genome, and then in the other organisms genomes. BLAST searches
can be performed at www.blast.ncbi.nlm.nih.gov. After the specific BLAST searches to obtain the
sequences are preformed, use the tab BLASTP to perform a protein sequence alignment. After the
alignment is preformed the percentages must be compared, and are used to find the amount of
similarities between the organisms and humans.
Results:
Organism Name: Percent Matched: Percent Gap:
Homo Sapien N/A N/A
Xenopus 90% 5%
Danio rerio 89% 5%
Mus Musculus 91% 7%
The first organism the Xenopus had a 90% alignment match, and had a 5% gap. The second
organism the Danio rerio had an 89% match and a 5% gap. On the Mus musculus the amount that
was a perfect match with the Homo sapiens was 91%, and the amount that was gaped was 7%.
Conclusion:
Arrestin as a protein is found in all four of the organisms. Homo sapiens and Mus musculus were
the two closes in terms of alignment and gaps. The hypothesis is held up through the alignment.
After looking at the lineage of the four organisms we can see that the Mus Musculus is the closes to
the Homo sapien because their lineage does not break at the Euteleostomi clade. Both the Xenopus
and the Danio rerio split from the other two and go into Amphibia and Actinopterygii

Bradford Assay Lab Report
Abstract For this lab we will use Bradford assay and spectrophotometers in order to determine the
concentration of protein in commercially produced food products. The experiment concluded the
three given milk samples, breakfast milk has the most amount of protein. After calculating and
recording data, we founded that whole milk had the least amount of protein, and breakfast milk
ended up having the most protein. Leaving Muscle Milk with less protein than the breakfast milk
and more protein than the whole milk. This research will help people in wisely choosing their intake
of commercially produced food.
Introduction
"Using protein and amino acid supplements include stimulation and maintenance of muscle growth
and strength, enhancement of energy ... Show more content on Helpwriting.net ...
First we began setting the spectrophotometer to 595 nm. We than label each cuvette with numbers
corresponding to the concentrations. To each cuvette we added 1 ml of coomassie blue dye and on
the blank cuvette we added 20 ul of PBS (Phosphate Buffered Saline). We then added 20 ul of each
standard to the respective cuvettes using a new clean tip for each use. Each cuvette was covered
with parafilm and inverted to mix allowing it to sit for a total of 5 minutes. We first inserted the
"blank" cuvette into the spectrophotometer and pressed blank once right after we put in each of the
cuvettes with the protein concentrations. In between each cuvette we put in a blank before the next
step. After the absorbance were recorded, we created the standard curve. We then graphed the
standard curve, with solutions and their protein concentrations. We then labeled each cuvette along
with a blank. We repeated the process from finding the standard curve with the known solution. We
then used the equation from the standard curve in order to find the concentrations of proteins and
compared. The concentration is the independent variable which is plotted on the x–axis and the
absorbance is the dependent variable which is plotted on the y–axis. For the mathematically a
straight line of best fit is used by using the point–slope equation, y = mx + b, where m is the slope of
the line and b is the

Methods And Methods Of Mouse Proteins
Methods and Materials
Mouse PrP would be used for this study since it is known that mouse PrP share a number of
structural and biochemical characters with mammalian PrP (Harreist et al. 1991; Gabriel et al.
1992). The materials and methods to be used are similar to those used by Agnes Lau et al. (2015)
Mutant PrP plasmid constructs
To create new alleles in the PrP which would be equivalent to the copied peptides, to be examined
by electron paramagnetic resonance (EPR) spectroscopy– also called electron spin resonance (ESR),
a technique used to study chemical species with unpaired electrons. EPR spectroscopy plays an
important role in the understanding of organic and inorganic radicals, transition metal complexes,
and some biomolecules –the sequences that encode the N–terminal portion of prion and part of the
5' untranslated region will be synthesized by GenScript Inc. This will eliminate novice mistakes on
my part. KpnI (from New England Biolabs), a restriction endonuclease enzyme will be used to
remove the encoding sequence of concern. T4, a DNA ligase will be used to anneal (recombine
DNA into a double–stranded form following separation) the full–length prion sequences into an
already existing pCDNA3 mammalian expression plasmid with the CMV promoter and a
neomycin–resistance marker (New England Biolabs), and correct full–length prion sequences will
be excised into another vector pBud egfp (Addgene) by digestion with XbaI and HindIII digestive
enzymes (Promega Biolabs).

What Is Type VI Secretion System?
"Aim, load, fire: the type VI secretion system, a bacterial nanoweapon." – a Summary –
–Maria Mitchell
T6SS, Type VI Secretion System, is a specialized protein secretion system used by bacteria for
survival. This system acts as a "gun" firing proteins into the target cell. It is one of 6 found in Gram
negative bacteria and has been known to target eukaryotic cells, as well as competing bacteria. T6SS
serves as a virulence factor towards its host while also effectively killing its competitors, responding
in stress and horizontally transfering DNA. Certain systems also recognize other cells as themselves.
T6SS injects effector proteins into target cells in a one–step process. 3 subunits (TssJ, TssL and
TssM) serve to anchor the system. ... Show more content on Helpwriting.net ...
T6SS is believed to resemble the tail of a bacteriophage. TssJ, TssL and TssM proteins combine to
form a complex which anchors it to the cell membrane, stretching from outer to inner membrane. A
baseplate, created by TssA, E, F, G and K resides within the inner membrane. VgrG resides within
the baseplate and supports the tube of Hcp hexamers extending into the cytoplasm. TssB and TssC
combine to form a sheath–like structure supporting the Hcp hexamer tube. Their interlacing
construction provides strength to the barrel during contraction and effector release. At the tip PAAR
and VgrG combine to create the "spike" at the end of the tube, which serves to puncture the
membrane of the target cell, so that the effector proteins can be delivered. PAAR repeat containing
proteins have been found to play an important role in the formation of the puncturing structure at the
top of the tube, as their omission in certain bacteria result in loss of virulence. Once the effector
proteins have been successfully delivered to the target cell, ClpV attaches to TssC to promote
disassembly of the TssBC sheath. A specific defensive strategy, termed 'Tit for tat' has been
described in Pseudomonas aeruginosa. This is made possible by TPP (threonine phosphorylation
pathway) accessory proteins during cell to cell connection. PpkA and PppA act upon Fha1.
Accessory proteins TagQ, R, S and T lead to a change in conformation and a firing event. All
accessory proteins are necessary for activation. This strategy is activated in response to aggression
from another cell. Not all P. aeruginosa organisms contain the necessary components for this defense

Importance Of Sequence Specific Primer PCR
Sequence specific primer PCR
With the advancement of sequencing technology, vast information on DNA sequences of many
animal genomes has been developed (Goff et al. 2002; Yu et al. 2002). In order to correlate DNA
sequence information with particular a phenotype of a trait, sequence–specific molecular marker
techniques have been designed. Expressed sequence tags (EST) analysis is one such type. By
adopting this method, Expressed sequence tags of many animal species have been created and these
sequences are subjected as putative functional genes by using advanced bioinformatics tools.
Allozyme markers
Allozymes are allelic variants of proteins produced by a single gene locus, and are of interest as
markers because polymorphism exists and ... Show more content on Helpwriting.net ...
They usually exhibit simple Mendelian inheritance and codominant expression, making genetic
interpretations easy. Disadvantages associated with allozymes include heterozygote deficiencies due
to null (enzymatically inactive) alleles and the amount and quality of tissue samples required (Hillis
et al., 1996). In addition, some changes in DNA sequence are masked at the protein level, reducing
the level of detectable variation. Some changes in nucleotide sequence do not change the encoded
polypeptide (silent substitutions), and some polypeptide changes do not alter the mobility of the
protein in an electrophoretic gel (synonymous substitutions). Moreover, genetic variations caused by
mutations are expressed as amino acid replacements due to changes in protein compositions, and are
resolved as bands (alleles) on electrophoresis (DeYoung and Honeycutt, 2005). However, other
limitations developed by allozymes are highly biased genomic sampling (only genes encoding well–
documented, soluble proteins are detectable); a low number of markers, insufficient for examining
major portions of the genome; occasional differences between tissues or ontogenetic stages; and
difficulty in the standardization of experimental methods from laboratory to laboratory (DeYoung
and Honeycutt, 2005). Low levels of genetic variation revealed in many allozyme studies of animal
populations prompted a continued search for markers with greater genetic resolution.

Exam 2 Answers Essay example
Biol 385: Biotech. and Gen. Eng. Name ___________________________
Exam #2 – 100pts 06 April 2012 G # _____________________
=======================================================================
If necessary, use the back of the exam pages for the rest of your answers. Do not use other sheets of
paper. Please write legibly; if I cannot read your answer, I will count it wrong. * BY TAKING THIS
EXAM, YOU HAVE AGREED TO ABIDE BY THE SPIRIT AND THE LETTER OF THE
HONOR CODE OF GEORGE MASON UNIVERSITY. INITIAL HERE
=======================================================================
1. (6pts) Expression of eukaryotic proteins in prokaryotic cells occasionally results in instability or
... Show more content on Helpwriting.net ...
Draw a schematic of the vector and include all of the genetic loci that will permit this two–gene
vector to function as a shuttle vector between human cells and E.coli cells. (For your convenience, I
have put the A and B polypeptide coding regions on the two–gene vector schematic.)
See Figure 7.20 on page 184, Chapter 07 of the text.
A two gene vector will have a two promoters and two polyA sites to permit expression of both
coding regions, which will be on two mRNAs. As a shuttle vector it will also have an E.coli ori and
selectable marker, and a eukaryotic selectable marker and either a eukaryotic ori or sequences to
permit homologous recombination into the chromosome.
7. (3pts) A technician from the BIOtique, Ltd. Protein Engineering Group comes to you for advice
regarding her protein of interest. She needs to improve the enzyme in order for Biotique, Ltd. to
compete effectively in a new commercial market area. What are three properties of her enzyme that
you could suggest that might be useful to change by directed mutagenesis in order to improve her
enzyme?
1) kinetic property – Vmax kinetic property – Km Thermal stability pH optimum
2) altered allosteric regulation increased resistance to degradation removal of co–factor requirement
increased reactivity in non–aqueous solvent
3) altered specificity of substrate binding
8. (6pts) BIOtique, Ltd.'s Molecular Genetic Diagnostics

What Is Western Blotting
This article delves into far western blotting which was obtained from the standard technique of
western blotting. Western blotting is a technique that is used to detect a protein immobilized on a
matrix. Furthermore, the steps in the standard and far western blot method are similar and consist of
preparing purified prey protein. It also consists of running an SDS–PAGE gel where the proteins are
transferred to the membrane and reduced and denatured with the Laemmli buffer. The final steps
include blocking or preparing purified bait proteins, then incubating the membrane with those
proteins, and finally detecting the proteins on the membrane with the western blot method.
Before starting this type of experiment, it is important to have an antibody that knows the protein
that will be worked with. The experiment used in this article used lysates of cultured mammalian
cells as well as purified bait proteins. To have ... Show more content on Helpwriting.net ...
Light was also created when revealed to horseradish peroxidase and this lead to the light being
shown on photographic film. Other detection methods include protein staining with Coomassie Blue
which for example, provides an image of the proteins shifted onto the membrane. In addition, it was
noted that failure may result if denatured proteins are unable to form interactions and can even result
with exchanges with nonspecific proteins by proteins in non–native conformations. Also that
denaturing likely affects prey proteins more. Moreover possible problems that could arise in this
method is that there could be incomplete transfer or even too many bands seen on the blot. The
solution for the latter would be to check the antibody with the western blot standard or to reduce
their concentration/incubation length. Taken as a whole the far western blot prospers when it
resembles the western blot

comparative proteomics Essay
Comparative Proteomics: Protein Profiler Lab by Jonathan Thulson
Biology 113
October 6, 2013
Lab Partner:
Vernon Morris
INTRODUCTION Proteomics is the study of proteins. Their functions, interactions with other
proteins, cellular locations and levels at which they are expressed. The purpose of this lab was to
compare the proteins present in different species of fish to be able to determine which species of fish
have the closest relation. This can be determined based on which two fish species have the most
proteins in common with one another. The Central Dogma of biology is a process in which a gene
made of DNA is transcribed by a messenger RNA and then translated into a protein. Based on the
Central Dogma of ... Show more content on Helpwriting.net ...
The proteins are also added to a Laemmli sample buffer in order to give each protein a negative
charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into
the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom
of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance
in which the proteins travel through the gel. If it is run for too long, the proteins will run off the
bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the
gel is stained in order to be able to see the individual bands of the different proteins. When the gel is
stained, the protein distances will be able to be measured and compared. For a detailed procedure,
refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.
RESULTS
I did not get conclusive data from the gel I made. As you can see in figure 1, the bands that showed
up on the gel were too cluttered to be able to measure them. So I could not compare protein bands
between the fish species based on our gel. Instead, I used a default gel picture that another group did
in the class to get my data. From their gel I was able to compare the different species. Table 1 shows
the number of bands that were similar between the different fish species when they were compared.
I was able to determine that fish species C (Tuna)

Chemotherapy Case Study
Background: A main challenge in chemotherapy is the delivery of an effective dose of a given
cytotoxic agent to the tumor site. Immunotoxins are protein–based drugs consist of a target–specific
binding domain with a cytotoxic domain to eliminate target cells. Such compounds are potentially
therapeutic against diseases such as cancer. The B–subunit of shiga toxin (STXB), which is nontoxic
and possess low immunogenicity, accurately binds to the globotriaosylceramide (GB3/CD77), that is
expressed in high amounts on a number of human tumors for example pancreatic, colon, and breast
cancer cells. Therefore, this toxin can be applied to target GB3–positive human tumors. In this
study, we evaluate a new antitumor candidate called DT–STXB chimeric ... Show more content on
Helpwriting.net ...
Key words: In silico modeling, diphtheria toxin, shiga like toxin part B, Cancer Immunotherapy.
Introduction
The patients with cancer, who is treated by surgery, chemotherapy and radiotherapy, the recurrence
usually happens and in many cases the tumor become metastatic(1, 2). For many years, the general
therapy of cancer was based on the nonspecific treatments with cytotoxic substances to kill
neoplastic cells. Yet, chemotherapy have some limitations, because it causes damage to natural
tissues and therefore the toxic doses must be confined(3). Because of confined remedial index of
cytotoxic drugs and the potential of cancer cells to develop resistance to these medications, it is
essential to improve the novel instruments to target aggressive malignancies alone, or in
combination with cytotoxic factors. Antitumor action of bacterial toxins have recognized in
numerous studies. The conjugated compound of bacterial toxins and the ligandhas assumed to kill
several kinds of tumor (4–8) . Thus in this study we have selected DT and STXB as an antitumor
agent. Native diphtheria toxin (DT) is a 538–amino acid protein containing three domains. Subunit
A is the enzymatically active domain, Subunit B has a hydrophobic domain at the N–terminal
portion, and the C–terminal portion of subunit B is the receptor–binding

Proteins : A Major Molecular Constituent Of Cells
Proteins are a major molecular constituent of cells they are vital for all living organisms and make
up about 15% of body mass. They are versatile macromolecules as they serve as a catalyst for
biochemical reactions (Hayward, 1999).
Amino acids are amphoteric and are the flat form of proteins. Only twenty occur in those proteins
found in humans (Nelson et al., 2008). Molecules called polymers link together in repeating units
and form to become a protein. Proteins frame structural and motor elements in the cell and each
protein and are the end product of the decoding process, which starts with the information in DNA
and has its own function determined by its gene (Alberts, 2002). Figure 1. Imagine showing the
twenty amino acids occurring in proteins (Particlesciences.com, 2015).
Proteins fall into two groups which are globular and fibrous. Globular proteins are mostly enzymes
which are high folded. Fibrous proteins however are typically structural (Chemguide.co.uk, 2015).
Their role is to give cells and tissues structural integrity and strength (Crowe and Bradshaw, 2014).
Protein chains arrange themselves to maximise the intro and intermolecular bonding. Various protein
shapes are folded into three dimensional structures into a conformation of lowest energy. The forces
driving protein folding are, hydrophobic groups, hydrophilic groups, charged side chains and
cysteine residues (Rich, 1955). Proteins can unfold and this process is called denaturation. This
happens at

Purified Protein Lab Report
Biotechnology industry, Research, Pharmaceutical industry used purified proteins in large quantities
for their purposes. This able genetic engineer to set up some technics to easily extracted from
variable source proteins in large amount.
Several approaches can be envisaged to address the function of a gene. The techniques of molecular
biology and biochemistry allowing for example to localize the expression of a gene or its product
(Northern, western, in situ hybridization, immunofluorescence, etc.), to determine the structure of
the protein (NMR, crystallography ...) or to find partners proteins (double hybrid,
immunoprecipitation ...) give important information on the function of a gene (V Ecochard –2011).
This experiment will focus on SDS PAGE of purified protein and cell lysate samples, Western blot
analysis procedure, interpretation of purified protein and lysate samples; Sequence analysis of
tagged fusion proteins, and why and how to choose cell type, and why cell expression mater? The
following samples containing human dihydrofolate reductase (DHFR) protein will be analyzed:
Purified His–tagged DHFR, expressed and purified from E.coli, ... Show more content on
Helpwriting.net ...
Place the gel in the electrophoresis chamber and fill chamber with with 1X running buffer, and
carefully rinse wells by pipetting to remove any bubbles.
Sample preparation: purified protein and cell lysate have been already and denatured in Laemmli
sample buffer and are in His–tagged DHFR, GST–tagged DHFR, Myc–Flag–tagged DHFR aliquots.
Control lysate is provided by TA. Wear goggles before to hreat samples for 2 minutes at 95˚C.
Centrifuge samples and load gel at room temperature. Obtain an aliquot of the each of the following:
1X Laemmli buffer and Kaleidoscope prestained protein standard (Bio–Rad

Total Serum Protein Determination Lab Report
Introduction
Total serum protein determination is a standard clinical test that is often used for the diagnosis of
diseases involving the kidneys, liver or bone marrow and other metabolic or nutritional disorders1.
Blood plasma contains approximately 7% proteins by weight and the fluid that remains after the
clotting factors are removed from the plasma is the serum2. Albumin and globulin are the most
common blood proteins, and the total protein is the combined measure of the two3. The most
abundant protein is albumin, which acts as a carrier of many different molecules in the blood and
helps maintain blood volume2. The globulin proteins include many enzymes and elements of the
immune system such as antibodies and immunoglobulins2. Changes in ... Show more content on
Helpwriting.net ...
This method is a chromogenic test that uses biuret reagent to detect the presence of peptide bonds in
blood serum12. The active ingredients in the reagent are sodium hydroxide, potassium sodium
tartrate and copper sulphate pentahydrate13. The development of a violet colour after incubation at
room temperature is indicative of the presence of proteins and peptides in the blood serum12. A
coloured coordination complex forms when the copper ions (Cu2+) binds to the deprotonated
nitrogen atoms that form the peptide bonds of the amino acids12. Reactions can also occur between
Cu2+ and the nitrogen in the amide groups and the imidazole rings12. The colour intensifies after a
period of incubation as the alkalinity of the solution increases and more deprotonated nitrogen atoms
become available12. The concentration of the protein and the colour intensity are proportional,
thereby exhibiting a linear relationship in accordance with the Beer–Lambert law14. Absorbance is
measured by spectrophotometer, λmax = 540

GFP Protein Lab Report
The purpose of this lab was to purify and test a GFP protein via several laboratory methods for the
purpose of purifying and testing the protein in SDS–PAGE. To purify the protein chromatography
and gel electrophoresis were the methods used in the experiment. GFP in the samples were tested
using an ultraviolet light. When GFP was found present the cell were transformed into a petri dish
containing ampicillin and arabinose. The cells were then lysed and SDS–PAGE was used to test.
The results from the SDS–PAGE were inconclusive revealing there was no GFP present in the
samples two through four. Introduction The purpose of this lab was to purify GFP protein using
different laboratory methods. GFP is a green fluorescent protein found in jellyfish ... Show more
content on Helpwriting.net ...
250 ul of supernatant was then transferred into a new tube and 250ul of binding buffer was then
added and the supernatant and pellet where then checked with a UV light to see if it glowed green.
The column was then run. 400 ul of lysate/binding buffer was added to the column. "Load" 100 ul.
The liquid that passed through the column was collected and labeled "Flow–through" 300 ul of wash
buffer was then added to the column. The liquid was then collected and labeled "Wash" 300 ul of
elution buffer was then added, the liquid was collected and labeled "Elute 1". This step was repeated
twice while collected the excess and labeling "Elute 2" and "Elute 3". Once the last elution ran
though the bottom of the tub was capped. 300 ul of elution buffer was added and mixed in with
beads to make slurry; the excess was collected and labeled "beads". The presence of GFP was then
checked by using a UV light, after checking for GFP the tubes were placed in a 4 degree Celsius
refrigerator. Begin with lab with the 7 tubes made from the previous lab, a blank with water, and a
set of 6 standards with known protein concentrations: 1 mg/ml, 0.5 mg/ml, 0.125 mg/ml, 0.0625
mg/ml, and 0.03125

Genetic Testing And Identification Of Dna Extraction
Introduction.
Extracting DNA from available cells is a widely used practice with applications such as forensics,
genetic testing and victim identification to name a few. DNA extractions have been widely practiced
for decades. This report simply summarises our results comparing two methods.
The tests compared the effectiveness of the often cited 'salting out method' based on Sunnucks and
Hales' (1996) method of DNA extraction from aphids, with a 'science–at–home' style method of
DNA extraction according to the NHGRI's video "how to extract DNA from strawberries" (2010)
This made use of household detergent as a surfactant to allow the DNA out of the cell and its
contents. This method is cheaper to run, not involving costlier laboratory chemicals like Proteinase
K. (Ciulla, Sklar and Hauser, 1988, page 488)
Each requires high precision to differentiate between samples from different individuals, and it is
ideal to produce accurate results from a small sample. The sample must consist only of DNA for gel
electrophoresis to give proper results, containing no protein or lipid impurities.
The samples must be pure to be comparable, something that the YouTube method cannot offer with
reliability due to not using Proteinase K, which was applied early in the salting out method to
denature proteins for later removal.
The YouTube method is predicted to obtain DNA, but not with the same consistency or accuracy as
the salting out method, due to the lack of time and

Food Protein Derived Bioactive Peptides ( Bps )
Abstract
Food protein–derived bioactive peptides (BPs) have been reported to trigger certain physiological
responses in the body, thereby influencing health positively. These peptides have attracted high
research and consumer interests due to their huge potential of use in functional foods and other
dietary interventions of disease control and health promotion. However, successful product
development is limited by the fact that current manufacturing processes are either difficult to scale
up, high in cost, or have the potential to affect the structure–activity properties of these peptides. To
overcome these challenges, we have proposed in this review, the use of 'omic ' techniques and high
throughput simulation models to forecast the ... Show more content on Helpwriting.net ...
Introduction
Over the years, it has become a known fact that health and nutrition are intricately linked. Not only
do food nutrients supply the necessary biomolecules for various metabolic activities, but, in some
cases, food nutrients are able to trigger certain desirable physiological responses in the body. Food
proteins and hydrolysates thereof are amongst the most well studied bioactive molecules (Danquah
and Agyei, 2012). Bioactive peptides have been defined as protein hydrolysates which, upon entry
and absorption into the body, have the ability to induce certain desirable and physiologically
measurable 'hormone–like' activities(Korhonen and Pihlanto, 2006). Some biological functions
induced by these peptides include immunomodulatory, cytomodulatory, opiate, antihypertensive,
antimicrobial, antithrombotic and metal–chelation activities (Möller et al. , 2008). As natural
products of food origin, bioactive peptides have a huge potential in health–promoting functional
foods and therapeutic products(Korhonen, 2009).However, this potential is not being realised as a
result of certain bioprocess challenges. The lack of commercially–viable processes for large–scale

The Protein Interactions Between Gst, Gst Ebna1 And 6xhis...
In this investigation, experiments were conducted to determine the protein–protein interactions
between GST, GST–EBNA1 and 6xHis USP7 through affinity chromatography, overexpression and
SDS PAGE analysis. The hypothesis states that the EBNA1 would interact with 6xHis USP7,
whereas the GST peptide would not be able to bind to 6xHis USP7. The results of the experiments
performed mostly supported the hypothesis, and are further discussed in this section.
Ni+2 affinity chromatography purifies 6xHis USP7 fusion protein by using a Ni2+ resin and an
imidazole ligand to elute the desired protein; in this method 6–9 histidine residues are used as the
tag. Imidazole was used in many different concentrations to disrupt the interaction between the His
residues and NI+2 also, higher concentrations are needed to elute which allows imidazole to
compete for binding to the resin with the His–tag. However, GST affinity chromatography purifies
GST and GST–EBNA. This method allows for the elution of GST by using a GSH resin and ligand.
It should also be noted that the GST tag acts as a substrate for the ligand to bind to immobilize the
affinity column(Bornhorst and Falke, 2004).
The concentration of the purified 6xHis USP7 was determined by using a Bradford Assay to create a
standard curve and to obtain a linear equation. The calculated concentration was 0.32mg/ml. This
value falls into the range presented on the graph, and thus ensuring the ability for the elution fraction
to undergo

The Rna Interference ( Rnai ) Pathway
The RNA interference (RNAi) pathway is an important biological system that is utilised by many
organisms as a method of regulating gene expression. But what exactly is RNAi? And what is the
importance of its use and application in biology?
RNAi is a cellular process that actively silences specific genes. This method of post–translational
gene regulation has been observed in many organisms including plants, fungi, some bacteria and
animals. RNAi inhibits protein synthesis of the target transcript by the use of small RNA molecules
that target messenger RNA (mRNA) within the cytoplasm following transcription by an RNA
polymerase. RNAi occurs during translation of the mRNA by inhibiting the action of Ribosomes
that catalyse this process within ... Show more content on Helpwriting.net ...
Upon binding the two strands of siRNA are separated, with one strand – known as the guide strand –
remaining bound to RISC and the other strand being degraded. The guide strand then directs RISC
towards its target mRNA for cleavage (Zamore, et al. 2000). This mechanism is extremely precise as
it relies on complimentary base pairing between the bound siRNA and its target mRNA. This
complementarity allows RISC to bind to the target mRNA where the Argonaute protein catalyses it's
cleavage, resulting in degradation of the target mRNA (Fig. 1).
This mechanism inhibits translation of the protein for which the target mRNA codes for and
therefore silences that specific gene. RNAi can also involve the use of coded short hairpin RNAs
(shRNAs), which are larger dsRNA molecules containing a loop section which folds back on itself
to form a double stranded molecule contains a sense and antisense strain. The shRNAs are cleaved
into siRNAs when they bind to RISC and can also found in examples of exogenous introduction
such as viral or experimental insertion. The ability to post translational control of gene expression
with a precise sequence specific manner has been exploited experimentally in researching gene
knockdown and loss of function gene analysis.
In 2006, Andrew Fire and Craig Mello were awarded the Nobel Prize in Physiology or Medicine for
their work in RNAi in Caenorhabditis elegens. Their 1998 paper demonstrated RNAi of endogenous

Structure And Function Discovery Of Proteins
CHAPTER 1
INTRODUCTION
PROTEIN SEQUENCE
Proteins (Y.Vincent, et al.) are present in every cell of the organisms. They are involved virtually
almost in all cellular activities. They are responsible for the various metabolic activities, nutrition
transportation, regulations and etc. They exist as single chain molecule, as a three dimensional
structures or even in the bundle or complex forms. The protein plays a vital role in cellular
processes. The protein consists of twenty amino acids. They possess different characteristics such as
hydrophobic, hydrophilic, polar, non–polar and etc. It is the great challenge to the bioinformatics
that to find which combination of proteins are responsible for what kind of activities. The structure
and function discovery of proteins in living organisms is vital role in understanding the background
of various cellular processes. It is helpful in treating various diseases, in detecting the drugs to
peculiar diseases. The IUPAC code for Protein from DNA code
The irregularity in the proteins can be found if their actual functionality and their protein complex
are known. The biologist will interpret the functionality of the protein complex based on their
chemical properties.
Amino acid codes
1–letter 3–letter Description
AAla Alanine
R Arg Arginine
N Asn Asparagine
D Asp Aspartic acid
C Cys Cysteine
Q Gln Glutamine
E Glu Glutamic acid
G Gly Glycine
H His Histidine
I Ile Isoleucine
L Leu Leucine
K Lys Lysine
M Met Methionine
F Phe

Protein Chromatography: A Concise Guide to Ion Exchange Chromatography

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Protein Chromatography: A Concise Guide to Ion Exchange Chromatography