Advantages And Disadvantages Of Protein

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 104ВєC 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
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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, ...
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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
95ЛљC. 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

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 ВµL of whole
bovine blood was transferred into a yellow microcentrifuge tube that was labeled WB using a P–200. 50 ВµL 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 ВµL of the supernatant from the Clear tube was transferred into a
yellow tube labeled WP and 50 ВµL 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 serialdilution 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 BradfordAssay. 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

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
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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

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

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 ...
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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, О»max = 540

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), NaГЇve 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–О±, all–ОІ, О±+ОІ, О±/ОІ. 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 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

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).

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
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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.

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
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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

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
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

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 totalprotein. All other chemicals
were obtained from reputed companies.
Animals and experimental design
Twenty male albino rats, weighing 90 В±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 (23В±2 CВє) 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

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
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 (MГ¶ller 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

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 (Оі) 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 (О± = 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 (О± = 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

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
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 500Ојl 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 200Ојl 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 500Ојl 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 30Ојl 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

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

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

. 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 ПЃ and Пѓ 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 (О”О”G)
in kilocalories per mole, due to the presence of a specific nsSNP (Pires et al., 2014). The difference in Gibbs Free Energy (О”О”G) 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

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

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

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 Helpwriting.net ...
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

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

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

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
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

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 aprotein. 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)

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 О±–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

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

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

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 SapienN/AN/A
Xenopus90%5%
Danio rerio89%5%
Mus Musculus91%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

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

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

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 Helpwriting.net ...
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

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–letter3–letterDescription
AAlaAlanine
RArgArginine
NAsnAsparagine
DAspAspartic acid
CCysCysteine
QGlnGlutamine
EGluGlutamic acid
GGlyGlycine
HHisHistidine
IIleIsoleucine
LLeuLeucine
KLysLysine

MMetMethionine
FPhe

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 IОєB–О± 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 content on Helpwriting.net ...
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

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

Exam 2 Answers Essay example
Biol 385: Biotech. and Gen. Eng.Name___________________________
Exam #2 – 100pts06 April 2012G #_____________________
=======================================================================
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

Advantages And Disadvantages Of Protein

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