The Rice Genome

The Rice Genome
The rice genome carries at least two active GA20ox genes namely GA20ox1 and GA20ox2. It was
found that GA20ox2 was located on the long arm of chromosome 1, tightly linked to the sd1 locus
whereas GA20ox1 does not correspond to the sd1 locus as it is mapped on chromosome 3 of rice
genome (Matsuoka et al., 2002).
As in figure 2.5, sd1 corresponds to GA20ox2 gene consisting of three exons and two introns. It is
strongly expressed in the leaf blade, stem and unopened flower, whereas GA20ox1 is predominantly
expressed in the unopened flower. Other GA20 oxidase which is encoded by GA20ox1 is
preferentially expressed in the reproductive organs which enable the flowers in sd1 plants to develop
and be fertilized normally. This explains how the plant height is reduced without affecting the seed
yield (Sasaki et al., 2002).
2.9 RNase P Ribonuclease P is a type of ribonuclease which cleaves RNA. Its function is to cleave
off an extra, or precursor, sequence of RNA on tRNA molecules (Stark et al., 1978). It is an essential
ubiquitous enzyme, present in all cells and cellular compartments that synthesize tRNA (Gopalan et
al., 2002). RNase P is a ribonucleoprotein complex and is responsible for the 5' maturation of tRNAs
(Frank and Pace, 1998). RNase P has been proposed as a novel RNA–based gene interference
strategy for down regulating gene expression. After the discovery of RNase P in E.coli, the
subsequent biochemical purification revealed that RNase P consisted of one protein
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Research Paper On Chromatography
Chromatography media selection
By Catherine Shaffer M.Sc.
Chromatography is a technique for separation of mixtures. The name combines the greek chroma, or
"color," with graphein, "to write." The technique was invented in 1900 by Mikhail Tsvet, who used
it "to write the colors" of plant pigments like chlorophyll carotenes, and xanthophylls. He did that by
dissolving the plant mixtures in a fluid, or mobile phase, and allowing it to flow through a solid
material called a stationary phase. The components of the mixture flow through the stationary phase
material at different rates, creating waves or bands. Originally, those bands were detected by visible
color differences. Detection methods are now based on the chemical and physical properties of the
molecules being separated, including but not limited to color, UV absorbance, size, charge, and
hydrophobicity.
In modern chromatographic separations, the stationary phase comes in the form of media, either
pre–packed in a chromatography column, or added by the user to the separation apparatus. The
choice of media depends on the type of molecule being separated, the means of separation, the
purpose and scale of separation, and the desired purity of the separated sample.
Generally, the material used in ... Show more content on Helpwriting.net ...
It separates proteins in a sample based on charge. If a negatively charged resin is used to capture
positively charged molecules, the method is referred to as cation exchange chromatography. The
opposite, in which the resin is positively charged and the target molecule is negatively charged, is
called anion exchange chromatography. An ion exchange resin is made using positively or
negatively charged functional groups on a solid matrix like cellulose, agarose, or polyacrylamide. A
protein sample is loaded onto the column in a buffer of low salt, and then flushed through the
column with a gradient of salt concentration or change in

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

Biomolecules Lab Report
Introduction
The genomic and plasmid DNA are extracted from Escherichia coli, and separated using agarose gel
electrophoresis in both non–diluted and 10–fold diluted forms. Charged biomolecules such as DNA,
RNA and proteins are separated using agarose gel electrophoresis due to the sieving properties of
the agarose. Smaller molecules requires a higher gel concentration with smaller microscopic pores
to act as a molecular sieve as they can sieve through faster than larger molecules. In this experiment,
the smaller sized plasmid is separated in the 1.0% agarose gel while the larger sized genomic DNA
is separated in the 0.7% agarose gel. The rate of migration of the biomolecules is also affected by
other factors including size, shape and charge of biomolecules, ... Show more content on
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Agarose gel electrophoresis result of genomic DNA in 0.7% agarose. Lane 1, Lambda HinIII–
cleaved DNA marker; Lane 4, 10X diluted genomic DNA; Lane 5, undiluted genomic DNA
Figure 2. Agarose gel electrophoresis result of plasmid DNA in 1.0% agarose. Lane 1, Lambda
HinIII–cleaved DNA marker; Lane 4, undiluted plasmid DNA; Lane 5, 10X diluted plasmid DNA
One fragment of genomic DNA is observed in the 0.7% agarose gel appearing in line with the first
band of the Lambda HinIII–cleaved DNA marker at 23kb. This result is consistent with the majority
of the groups.
Several fragments of plasmid DNA are observed in the 1.0% agarose gel. In the undiluted sample,
sizes of plasmid DNA are approximately 23kb, 9.7kb, 5.6kb and 2.5kb. In addition, there is a very
faint band at size 80kb. In the 10X diluted sample, however, have only 2 bands at approximately
16kb and 6.5kb. The undiluted sample result is most similar to result in lane 2 with 4 bands as well,
whereas the diluted sample is similar to the majority. Overall, majority of the bands indicated that
the plasmid DNA have 4 fragments.
In both gels, the undiluted DNA sample have evidently brighter bands compared to the 10X diluted
DNA

Acetone Milk Lab
Utilizing Acetone Powder of Several Organs to Compare Specific Proteins and Measure L–Lactate
Dehydrogenase Activity Abstract Animal organs contain a diverse amount of proteins that define
their function. In this study, acetone powder solutions of calf liver, bovine pancreas, chicken muscle,
porcine kidney, porcine brain, and porcine heart were analyzed through an SDS–Page gel to
determine diversity of proteins and through an LDH assay to determine enzymatic activity. It was
found that chicken breast muscle contained the most diverse collection of proteins along with the
highest LDH activity. This made reasonable sense considering that the chicken breast muscle is
composed of many different classes of actin and myosin proteins and that ... Show more content on
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The chicken breast muscle had the highest number of proteins present (13), while the porcine kidney
had the least (3) amount present (Table 2). The protein with the largest mass appeared to be the first
protein in the chicken breast muscle lane at 192.5 kDa (Table 2). There was also a trend that two
protein bands with an apparent mass of 64.6 ± 2.67 kDa and 78.5 ± 2.13 kDa appeared in each of the
different acetone powder lanes (Table 2). The protein with the smallest mass was found in the calf
liver at 17.0 kDa (Table

Gel Filtration Lab Report
Gel Filtration and SDS–PAGE of a Mixture OBJECTIVE. I This Lab Report is an analysis of the
results of a two–part experiment. In the first part, we used a gel filtration column to separate the
components of a mixture composed of protein and non–protein molecules. By doing so we hoped to
obtain fractions that contained single components of the mixture, while also gaining insight into the
relative molecular weight of each component compared to each other. We would then plot these
fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second
part, we would use the fractions which we had determined had protein to conduct an SDS–PAGE.
By doing so we hoped to determine an estimate on the molecular weight of the proteins present in
each fraction by comparing it to a tracker dye composed of a variety of molecules of differing
molecular weight. METHODS. II In the gel filtration step, we began with a slurry of Bio–Gel P–100
beads suspended in 20 mM phosphate buffer (Equilibration buffer) and an upright column with a
stopcock. We added enough of the Bio–Gel P–100 beads to the column until we reached a height of
4.7 cm, making sure not to allow the column to run dry and that the top of the beads column is flat.
We then added the sample, a mixture of Blue Dextran (2 mg/ml, 2 MDa), Hemoglobin (2 mg/ml),
Bovine Serum Albumin (2 mg/ml), and Yellow Food Coloring (5 μl/ml, ~500 Da) (Sheffield, 37).
This was followed by another ~1 ml of equilibration

Gel Electrophoresis Lab
Abstract The purpose of this study was to study the plasma membranes of erythrocytes of the human
and determine how the majority of polypeptides work with it and where they are located with
respect to it. The purification of the erythrocytes was executed by osmotic hemolysis and were
washed to the point of being "ghosts". The polypeptides were then analyzed on the basis of mass by
polyacrylamide gel electrophoresis; sodium dodecyl sulfate (SDS) was used to denature the proteins
and separate them from the lipids. Furthermore, the location of the membrane proteins were
determined by altering the environment around the membrane and recording how they responded.
The major conclusion obtained from this study was that the distribution of proteins along the inside
and outside of the plasma membranes of erythrocytes exhibits absolute asymmetry.
Answers to Questions ... Show more content on Helpwriting.net ...
In this experiment, the erythrocyte membranes were dissolved in sodium dodecyl sulfate (SDS),
which subsequently led the proteins to resultantly denature and separate from the lipids. The
samples are then placed in the apparatus which carries out electrophoresis; the mixtures are subject
to an electric field (the SDS places a greatly negative charge on the polypeptides, making their
intrinsic charge irrelevant) and the lighter ones migrate faster than heavier ones. Subsequently,
Coomassie blue is added so that the polypeptides can be observed on the polyacrylamide gel (blue
lines will indicate the polypeptides). However, some components (the heavily glycosylated
glycoproteins) of the experiment could not be stained through Coomassie blue or through other
normally used protein staining techniques; luckily, they can still be made visible by the periodic
acid–Schiff (PAS) staining

Western Blot Analysis
The technique used in this experiment was western blot to determine the protein levels in different
cow's stages; fetal calf serum, newborn calf serum and cow serum. Western blot is technique
commonly used to identify proteins by its movement in the gel electrophoresis. Western blot is use
to separate protein based on its molecular weight in gel electrophoresis. The proteins separated in
the gel, then transferred to a nitrocellulose membrane using an electron current (1). Finally the
membrane is incubated with proteins that would stain with antibodies specific for the wanted
protein. SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is used in this
experiment to detect the presence of antibodies in different serum samples

The Use Of Sodium Dodecyl Sulphate ( Sds ) Polyacrylamide...
Feedback/Response
The overall feedback was to state the aim and conclusion clearly, include more in–text citations and
references and be more detailed in elements of experimental errors. This feedback was taken into
account, as I have included more in–text citations and references, expanded on experimental errors
in more detail and ensured my aim and conclusion is clear.
Abstract
The purpose of this investigation was to identify the class of immunoglobulins using, sodium
dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE); by making deductions about
the structure and molecular organisation of the protein. The experiment was conducted to calculate
the unknown molecular mass for reduced and non–reduced immunoglobulin, using SDS–PAGE by
measuring the distance migrated. Even though the method is intrinsically inaccurate, it's enough to
deduct the class of immunoglobulin using Mr The experiment displayed that the distance migrated
decreased as the molecular mass (Mr) increased. Based on the number of bands visible on the gel, it
was concluded that sample A was reduced with 2 bands; heavy chain with Mr of 52.939 kDa and
light chain with Mr of 23.470 kDa. Whereas the B sample was non–reduced, with a total molecular
mass of 152.415 kDa. This illustrates that both reduced and non–reduced immunoglobulins are of
class IgG.
Introduction
The SDS–PAGE technique is used to identify immunoglobulins. As Shi and Jackowski (1998)
explain; electrophoresis is a process of

Invertase Lab Report
This experiment was conducted as per the BCHM 310 Laboratory Manual [3]. The first objective of
this experiment was to analyze the purity of the invertase fractions collected during experiment 6,
and to determine the molecular weight of LDH–H4, LDH–M4 and invertase subunits. This was
accomplished using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). In
this procedure, SDS, a negatively charged amphipathic molecule, was used to denature the proteins
and to give each protein a similar charge–to–mass ratio [4]. As a result, most oligomeric proteins
separated into individual subunits, and each subunit assumed a rod–like shape [4]. The distance
travelled by each subunit, along the polyacrylamide gel, was a function of its molecular weight;
where proteins with a greater molecular weight moved a smaller distance than proteins with lower
weights [5]. Since SDS is not a reducing agent, and no other reducing agent was added, oligomers
with disulfide bonds between subunits would have remained intact [4]. However, this was not
expected to be problematic for analyzing invertase or LDH isozymes, as these proteins lack
disulfide interactions between their subunits [2,6]. In addition, since invertase and LDH are homo–
oligomers, each protein's subunits were expected to migrate the same distance [2,6].
The proteins were visualized using the Coomassie Blue stain. Coomassie Blue binds non–
specifically and nearly stoichiometrically to all proteins [5]. Proteins have a higher affinity to the
dye than the polyacrylamide gel; therefore, after removing excess stain, the protein bands can be
visualized [4]. This non–selective binding was essential for analyzing the purity in each invertase
fraction, as both non–target proteins and invertase were visualized. As shown in Fig. 1., excluding
invertase fraction 2, the general trend was that the number of protein bands decreased from fractions
1 to 4, while a band with a molecular weight around 1.2 x 102 kDa became more prominent and
intense. These results were expected for a successful purification [4]. During experiment 6, it was
observed that the specific activity increased during each successive fraction. Since the same amount
of protein, from each invertase

P-Nitrophenol Enzyme Lab Report
The influence of changing amino acids sequence to the Alkaline Phosphatase (AP) enzyme optimal
functions
Abstract
Hydroxyapatite in the bone is regulated by an enzyme called alkaline phosphatase. If a mutation in
the AP enzyme exists, a possible outcome can be a decreased level of bone density. Through various
experimentations, the functions of normal and mutant AP enzymes were observed. The variations in
function of the enzyme were determined through different pH levels. In order to accomplish this, the
activity of alkaline phosphatase needed to be examined by assessing how fast the product p–
nitrophenol was forming. By incorporating the spectrophotometer, the optimal wavelength of light
to use in the AP experiment had to be determined by ... Show more content on Helpwriting.net ...
P–nitrophenol was measured at various wavelengths through a spectrophotometer to find the
optimal absorption. Once determined, p–nitrophenol was diluted in a buffer, NAOH, and PNP buffer
to establish the standard curve for p–nitrophenol and the extinction coefficient. A time course was
then established to determine over what length of time the reaction should occur. Substrate PNP and
the pH buffer 10 were added to test tubes and placed in the incubator at 5 minute increments. With
the information obtained through the experimentation, the hypothesis was ready to be tested. The
amount of time it took the AP enzyme to convert PNP to p–nitrophenol was recorded to determine if
there was a difference in the effect of pH on normal and mutant AP in heterozygous individuals. To
ensure validity of the functional differences between normal AP and mutant AP, polyacrylamide gel
electrophoresis was used. The center lane of gel with a pre stained protein standard was loaded first.
Homozygous normal AP extract (15ul), heterozygous AP extract (15ul), and purified mutant AP
(15ul) were the other three samples loaded into the wells. After the samples ran far enough, they
were transferred to staining trays and washed with 50 ml of gel wash buffer, AP buffer, and pH 6.
Next 50 ml of carbonate buffer, pH10 was added to one tray and 50 ml of citrate, pH6 was added to
the other tray. Lastly 1 ml of AP dye and substrate was added to each tray and placed in 37 degrees
Celsius water to incubate. Once the incubation was complete 50 ml of PBS was added to wash off
the excess staining solution and the gels were ready to be

Green Fluorescent Protein Chromatography Lab Report
Purpose: Green fluorescent protein (gfp) and blue fluorescent protein (bfp) extracted from bacteria
were separated by chromatography by means of a molecular sieve matrix. This process utilized the
proteins size and shape and was able to separate it from the other molecules and be collected in test
tubes. The tubes containing the desired protein will glow under U.V. light. By running an
electrophoresis gel with these proteins, one can compare the protein bands and be able to determine
the approximate molecular weight of the proteins. In this experiment, students learned about the
methods and procedures to partially purify gfp and bfp. After purifying the proteins, the students
will be able to compare the molecular weights of the samples by running a denatured SDS
polyacrylamide gel (Purification & Size Determination of GFP & BFP, EDVOTEK). This will show
students which proteins different from one another and how denaturing can affect the proteins.
Background: ... Show more content on Helpwriting.net ...
When energy is transferred to these proteins it causes them to emit light. They will also glow when
under U.V. light (Purification & Size Determination of GFP & BFP, EDVOTEK). The proteins are
extracted by using size–exclusion chromatography. This is a method where a solution is prepared
and separates molecules based on their size. The fluorescent properties of these proteins make them
heavily sought after when making chimeric proteins. Scientists will fuse bfp or gfp to another
protein to use it as a biomarker. These fusions will typically not affect the biological activity of the
chimeric protein (Purification & Size Determination of GFP & BFP, EDVOTEK). This makes
fluorescent proteins a tool that allows science to locate specific proteins within a cell and what their
purpose

RNA Induced Silencing Complex
The RNA–induced–silencing complex is used for gene regulation and in defense against viral and
bacterial infections. The RISC is loaded with small dsRNAs that will guide the complex through
base–pairing interactions to specific messenger RNA targets, which the RISC will help degrade.
Once degraded, no more protein can be made from these mRNAs. The RNA–induced silencing
complex can silence targeted genes through several mechanisms. It can work to repress translation
during protein synthesis, interfere with the transcript level through mRNA degradation, or work at
the level of the genome itself and eliminate DNA/form degrading materials. Heterochromatin is a
degrading material that can be created to modify or suppress genes in the RISC (MacRae ... Show
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Enzyme assays are laboratory methods used to measure enzymatic activity. They are often used in
the study of enzyme kinetics and enzyme inhibition. For our in vitro enzymatic assay, the RNA
substrate (a stem–loop RNA containing a 24 base pair stem) was mixed with either the wild–type or
mutant protein and incubated and room temperature. Incubating at room temperature produced a
delay in the expression of the proteins. To begin the catalysis, MgCl2 was added to the enzymatic
reaction. Magnesium is needed for catalysis in over three hundred enzymes including all of those
that utilize or synthesize adenine triphosphate (Ninfa, Ballou, & Benore, 2010). To stop the catalysis
reaction, EDTA and urea were added. Ethylenediaminetetraacetic acid (EDTA) will bind and remove
metal cofactors. In this experiment it neutralized the magnesium ion. Urea denatures proteins by
forming hydrogen bonds with protein backbones that are necessary to make up the structure of the
protein. Urea was added to our mixture to denature the wild–type and mutant

Driftless Area
I live in part of the upper Mid–West known as the Driftless area. The terrain contains high bluffs and
hills with narrow valleys. The Driftless area, unlike the rest of the Mid–West, escaped the terrain
smoothing, mineral depositing influences of the ice sheets during the glacial period (Jefferson,
2010). The bedrock contains limestone, sandstone and shale, and is exposed or near the surface
making it popular for sand mining. About 95 present of the frac sand mining in the United State is
done here. Driftless Area When the sandstone layer in the bedrock is broken down and crushed into
individual granules it is called silica sand. This type of sand is very uniform, non–porous and strong;
it can maintain its shape when exposed ... Show more content on Helpwriting.net ...
This material is often stockpiled separately on the site for use during mine reclamation (Wisconsin).
Unfortunately the wild life is displaced, bird migration is interrupted and the vegetation is destroyed.
The Driftless area has a lot of plants that are unique to this area only. Explosives are then used to
break the bedrock apart. The unwanted rock such as the limestone and shale are stock piled to be
used later for the reclamation process. Excavation equipment and dump trucks bring the sandstone
to the crusher that is located on the mining site. The crusher breaks the sandstone chunks into
individual sand grains. The sand has to be filtered to remove the smaller grains of sand that are not
acceptable for fracking. The entire process causes a release of silica dust. Inhaling silica dust can
lead to serious, sometimes fatal illnesses including silicosis, lung cancer, tuberculosis (in those with
silicosis), and chronic obstructive pulmonary disease (COPD). Silica exposure has also been linked
to renal disease and other cancers. In 1996, the World Health Organization – International Agency
on Cancer Research (IARC) identified silica dust as a "known human carcinogen" (Center). Due to
this risk factor "the federal government regulates workers exposed to silica dust, but there are no
limits set for the general public" (Wisconsin). This is a big

Essay On Hydrocarbons
Effective and economical production of hydrocarbons has gained great attention worldwide due to
the increasing demand for oil and the challenges oil companies face to deliver more oil from their
oilfields. Water flood is generally used as an economic and effective method in secondary mode
recovery after primary (natural) driving mechanisms have been depleted. Generally, water flooding
still leaves behind 60–70% of the oil initially in place (OIIP) inside the reservoir and Enhanced Oil
Recovery (EOR) methods are introduced to produce further oil from the reservoir. EOR is simply
defined as the injection of materials that do not naturally exist under the earth's surface to increase
the oil production rate of a reservoir. Primarily, there are ... Show more content on Helpwriting.net
...
The addition of alkaline increase the operation efficiency by controlling the adsorption between the
rock formation and chemicals present. The injection of alkaline also generates surface acting agents
when the interaction to certain types of oils in the reservoir occurs (Hill et all, 1973). It is also well
documented that the heavy and more viscous crude oils are the most responsive to alkaline flooding.
Objective of Study
The aim of the final project is to develop a model based on meta–analysis of available core flooding
dataset, thus, improving the understanding and underlying mechanisms of chemical EOR. This
required extensive and sophisticated analysis of theoretical background.
Summarizing all the above, the main objectives of this review is as follows:
Study all the evidence supporting chemical flooding in carbonate core flooding experiments. Core
flood experiments give an exact gauge of the oil activation potential through the injection of the
diverse chemicals. The procedure is however exceptionally confounded and can take a very long
time to accurately and successfully complete a solitary core sample, thusly; this paper will be
restricted to published core flood dataset only.
Identify the underlying mechanisms that have been suggested to support chemical flooding and
those that are not in support.
Report all the fundamental issues for the lack of understanding of chemical EOR.
Study the differences in underlying

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)

Gel Electrophoresis Lab Report
Gel electrophoresis is a simple technique that allows us to determine the charges and molecular
weights of all sorts of macromolecules. The basic theory is a simple one: more negatively charged
molecules will migrate in an electric field, toward the positively charged cathode. A matrix (such as
agarose or polyacrylamide) must be used to conduct heat evenly and provide an extra sieving effect.
In particular, agarose gel electrophoresis is generally used to separate DNA (single–stranded,
double–stranded, and supercoiled) and RNA. Since DNA is negatively charged, it migrates in an
electric field toward the positively charged cathode. The agarose matrix retards DNA migration
roughly proportionally to DNA length. Longer oligonucleotides have a harder time traveling through
the matrix, while shorter oligonucleotides breeze right through it. The concentration of agarose in
the gel can be fine–tuned to achieve optimal separation for a specific range of sizes. The higher the
concentration of the gel, the lower the pore size will be. So, when the target DNA size is small,
higher concentration of agarose gel is used. Usually 1 to 2% gels are used for detecting plasmids
(several kb long) or their fragments (i.e. from digestions). For ... Show more content on
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This buffer contains chaotropic salts which remove all DNA binding proteins and nonspecific DNA
strands from PCR products. This is done through disruption of protein structure by destabilizing
hydrophobic interactions. High salt existence decreases the negativity of DNA and increases binding
affinity and stronger interaction with the column membrane. Ethanol added washing buffer
efficiently removes dNTPs, primer dimers, salt, buffer or anything except DNA from the column.
For complete removal of residual ethanol, extra centrifugation is done. Finally, DNA is eluted
utilizing nuclease free water as binding affinity of DNA with nuclease free water is

Title – Gel Filtration and SDS Polyacrylamide Gel Electrophoresis (P.A.G.E) Gel Filtration, SDS–
PAGE Objective – The purpose of this lab was to separate a mixture of Hemoglobin, BSA, Blue
Dextran, and yellow food coloring by size into fractions using gel filtration. Also simple non–
quantitative assay was performed to determine the fractions that contained proteins. Another
objective was to analyze the protein content of being BSA or Hemoglobin and estimate the
molecular weight of the proteins collected. Methods – Gel Filtration: A glass column with inner
diameter of 1 cm, was packed with Bio–Gel P–100 (BIO–RAD) beads suspended in equilibrium
buffer of 20 mM phosphate buffer with a pH of 7.0 – 7.4. The height of the total content was kept at
5cm. The sample mix with total volume of .1ml contained 2 mg/ml Blue Dextran (blue, 2 MDa), 5
µl yellow food coloring (yellow, ~500 Da), 2 mg/ml Hemoglobin (red), 2 mg/ml of BSA in
phosphate buffer. As soon as the sample mix was added to the column, 0.25 ml (~2 drops) of
fractions were collected in the Eppendorf tubes until all the colored bands eluted the column
because it was assumed that the colored band represented protein content. Approximately 1 µl of
each of the fractions collected were spotted on to nitrocellulose filter using capillary pipet. After the
filter was dried, it was incubated in 50% methanol for 5 minutes then it was incubated in Amido
Black (0.1% amido black 10B in 30% methanol) for 5 minutes. Next, the filter was

Case Study : Integrative Study Of Cancer Cells
Integrative Study of Ligand Density, MMP Activity and Matrix Stiffness on 2D Substrates
Introduction Cancer cells' ability to migrate and metastasize to other organs and tissues in the body
is primarily responsible for disease mortality. Cell migration is caused by a cascade of biophysical
processes that interact to give rise to metastatic behavior. Four key biophysical properties involved
in cell migration are adhesion, contractility, cytoskeletal polymerization and matrix degradation.
Independently a lot is known about the cross talk between each of these biophysical process and the
extracellular matrix. Previous studies have shown that varying ligand density has a biphasic increase
then decrease effect on the speed of cell migration. It ... Show more content on Helpwriting.net ...
The stiffness I will use are 0.2kPa, 0.48kPa, 1.10kPa, 1.37kPa, and 1.67kPa to mimic the stiffness of
matrices in vivo. To visualize the cells deforming the matrix, florescent microspheres will be
incorporated into the matrix and traction force microscopy will be used to quantify the substrate
deformation caused by the cells migrating on the 2D substrate. Ligand density will be varied by
coating the polyacrylamide gels with type 1 rat tail collagen at varying concentrations 0.075 mg/ml,
0.15 mg/ml, 0.2 mg/ml, 0.4 mg/ml, 0.8 mg/ml. MMP activity will be varied using a broad spectrum
MMP inhibitor marimastat at 5μM, 10μM, 15μM, 20μM and 25μM concentrations in PBS. Since the
inhibitor is kept in a stock with DMSO I will also conducted a vehicle study to ensure the cells are
not affected by the DMSO. Time–lapse images of the cells migrating on the substrate will be taken
over 1hr 30min at 2 min increments using the florescent microscope available in Fraley Lab (figure
2). Cell migration will be quantified using metamorph software with the metavue track objects
software. The data taken of x and y positions for each 1min 30sec time point will be used to find the
average single cell velocity by finding the average of the distances traveled in each 1min 30sec time
point. Invasion distance will found by finding the displacement of the cell from the difference
between the x and y coordinates of the

Liver Kidney Disease : A Severe Form Of Hereditary Disease
Polycystic kidney disease (PKD) is a severe form of hereditary disease which results from a
monogenic disorder. The disease causes enlargement alongside with functional impairment. It
results in clusters of cysts mainly within the kidneys. Cysts are noncancerous round sacs consisting
of water like fluid which vary in size depending on its accumulating growth (Mayo Clinic Staff,
2014). This cystic disease consists of two forms, an autosomal dominant PKD (ADPKD) and an
autosomal recessive PKD (ARPKD). The purpose of this research paper is to increase chances of
healthy babies born to couples whom both transmit a mutation of PKHD1 gene, wanting to have a
child unaffected with autosomal recessive polycystic kidney disease (ARPKD). A recessive
mutation/disease is when the gene is inherited from both parents. This disease is of major interest
due to the amount of individuals affected widely. Over 12.5 million individuals have PKD, with
approximately 30% of affected children dying within their first year of life; making it a major source
of morbidity and mortality concern (Miyazaki et al., 2015). ADPKD makes up to 96–98% of all
cases, and ARPKD up to 2–5%. Although ADPKD is more harmful, the main focal point of this
research paper is on autosomal recessive polycystic kidney disease (ARPKD). Due to the
abnormalities in the biliary plates it leads to congenital hepatic fibrosis in infants. Congenital
hepatic fibrosis is the disease of the liver which is usually present from birth.

Sanger Sequencing: Polymerase Chain Analysis
Figure 1.1: A schematic representation of Sanger sequencing. The Sanger method uses
dideoxynucleotides that terminate newly synthesized DNA fragments at specific bases either A, T, C
or G. Then, the resulting fragments are resolved by electrophoresis through a denaturing
polyacrylamide gel in four parallel lanes, and the DNA sequence can be read (Rosenberg and
Pascual, 2014).
The initial Sanger sequencing method has been administered to several significant improvements
and developed remarkably over three decades. Even though cloning of DNA fragments into plasmid
vector was required in Sanger sequencing, the application of polymerase chain reaction (PCR)
(Saiki et al., 1988) for the amplification of specific DNA fragment in vitro has extensively

Poyacrylamide Essay
Polyacrylamide– This ingredient is not toxic, but when ingested, it breaks down and turns into
acrylamide, which is a carcinogen. The research that has been conducted on lab rats have found that
acrylamide do pose a risk of causing several kinds of cancer. The research on the effects on humans
has not yet been completed. The National Toxicology Program and the International Agency for
Research on Cancer consider acrylamide to be a "probable human carcinogen." This conclusion is
based on the data collected by the laboratory studies on animals. Even though the human studies are
not yet complete, I would still not use any product that contains any polyacrylamide.
Silicone–derived emollients– That's right, silicone–the same chemical that's used in breast implants.
This can cover the skin ... Show more content on Helpwriting.net ...
Caustic soda is also used in many oven cleaners. Sodium hydroxide is an ingredient in many
toothpastes as a pH stabilizer and skin creams. With contact on skin, this could cause contact
dermatitis.
This is information direct from the Sodium Hydroxide Material Safety Data Sheet (MSDS):
"POISON! DANGER! CORROSIVE. MAY BE FATAL IF SWALLOWED. HARMFUL IF
INHALED. CAUSES BURNS TO ANY AREA OF CONTACT. REACTS WITH WATER, ACIDS
AND OTHER MATERIALS.
Ingestion: Corrosive! Swallowing may cause severe burns of mouth, throat, and stomach. Severe
scarring of tissue and death may result. Symptoms may include bleeding, vomiting, diarrhea, fall in
blood pressure. Damage may appears days after exposure.
Skin Contact: Corrosive! Contact with skin can cause irritation or severe burns and scarring with
greater exposures.
Eye Contact: Corrosive! Causes irritation of eyes, and with greater exposure, it can cause burns that
may result in permanent impairment of vision, even

Macromolecular Analysis
The separation of macromolecules for laboratory analysis can involve the use of any of several
techniques that isolate macromolecular components on the basis of size, solubility, reactivity, and
volatility. Electrophoresis, for example, is a method of macromolecular separation that involves the
use of a suspending medium, known as the matrix, and an applied electrical current that separates
molecular components based on varying size and reactivity. While a uniform electrical current may
activate the movement of molecular components through the matrix, the sponge–like structure of gel
matrixes typically used in nucleic acid electrophoresis, such as agarose or polyacrylamide, inhibits
the uniform migration of these components through the matrix ... Show more content on
Helpwriting.net ...
After the macromolecular material has been introduced to the selected matrix within a controlled
apparatus, essentially a shallow plastic box, electrical nodes attached to opposite ends of the
apparatus supply the matrix with a positive and negative electrical current, respectively. Because of
phosphate groups present in nucleic acids, DNA and RNA possess a natural negative charge and will
be opposed by the applied negative current. The macromolecules will then begin to migrate away
from the negative node and toward the positive node along a horizontal pathway. As the
macromolecules migrate, their varying components will be caught and separated within the pores of
the charged matrix. In this sense, the matrix acts as a sieve rendering the components discrete for
analysis. Proteins, moreover, behave identically to nucleic acids during electrophoresis in terms of
reactivity, but require treatment with sodium dodecyl sulfate prior to electrophoresis. This detergent
binds with polypeptide chains within the protein and imparts it an overall negative charge. The
protein is then opposed by the negative charge and migrates toward the positive charge within the
apparatus. Multiple samples may be tested simultaneously within the electrophoresis apparatus
because each box contains small buffer lanes or "wells" that separate the samples within the matrix.
This allows for

Photosynthesis Lab Report
To prepare a Ni2+ –NTA–agarose resin column, 5 mL of deionized water and 1.5 mL of Ni2+ –NTA
resin slurry (Clontech, His60 Ni Superflow Resin) were added to a glass column. After thorough
mixing of the column and resin settling, the water was drained from the column until it reached the
top of the resin bed. 10 mL of extraction buffer (50mM NaH2PO4 (pH 7.5), 0.3 NaCl) was added to
the column and half of the volume was drained. The column was kept at 4 degrees Celsius until use.
Separation of Fumarase via affinity chromatography The affinity chromatography system consisting
of a Pharmacia P–1 peristaltic pump fraction collector, and Ni2+ –NTA resin column were attached
to a flow adapter. Crude yeast extract (6.41 mL) was loaded into column followed by a wash with 10
column volumes of extraction buffer. 5 mL fractions were collected at a flow rate of 1.54 mL/min.
Absorbance readings were obtained for each fraction using Simple Reads program on the Cary 50
UV–Vis spectrophotometer at 280 nm. Once Absorbance280 reached 0.05 or below to insure
sufficient washing of the column, 5 column volumes of imidazole elution ... Show more content on
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The prepared samples were heated with 12.5% (v/v) β–mercaptoethanol for 5 minutes in a boiling
water bath to denature the proteins. The SDS–polyacrylamide gel (Biorad, Any kD, Mini–
PROTEAN TGX Gel, 10 well, 30 µl) was loaded with heated protein samples of crude extract, flow
through, pre–dialysis, post dialysis, and molecular weight standard (Biorad). The gel was run at 24
mA until the tracking dye reached the bottom of the gel. The gel was removed and placed within a
stain (0.4 mg/mL Coomassie Blue R250, 40% (v/v) methanol, 5% (v/v) acetic acid) until bands were

Proteins Of Bovine Red Blood Cell
Protein Composition in Red Blood Cells in Humans using Polyacrylamide Gel Electrophoresis
Name: Emma Claypole
Date: Wednesday March 16, 2016
Lab Group: W08, Wednesday morning
2
Abstract
The proteins of Bovine red blood cell (RBC) membranes were analyzed using polyacrylamide gel
electrophoresis. After analyzing Bovine RBC they were then compared to human RBC counterpart.
Following finding the log of each molecular weight of each band, band one showed the highest
molecular weight. All five bands viewed were from humans. There are typically 7 bands visible
however, in our case only five were visible due to implications within the gel sample.
Introduction
Proteins are important elements in cellular membranes and give the membranes many of their
characteristics. In red blood cells, the meshwork of proteins in and around the membrane gives it
strength and flexibility, allowing a cell to squeeze through small capillaries without bursting. Other
proteins play roles in transporting material in and out of the cell (Lab Manual, Cell Biology).
Polyacrylamide gel electrophoresis (PAGE), with all of its different modifications is probably the
most widely–utilized procedure in contemporary biochemistry and molecular biology (Mordacq and
Ellington (1994)). In this experiment, we will attempt to determine the molecular weights of the
major proteins in the plasma membranes of bovine red blood cells (RBCs). The predictions made
are if our protein has similar weights as proteins

Rhyme Case Study
3.2.1. Creation of the reporter plasmid with VDREs
WT and mutant VDREs fused to a minimal promoter were ordered as premade G blocks with
restriction sites to allow cloning upstream of Firefly luciferase (Method xxxx). 3.2.2. Sequential
digest of SacI and BglII
As there is no buffer in which both SacI and BglII exhibit >50% activity, a sequential digest was
necessary (Method 2.3.). The digest was successful and the resulting cells were then lysed.
3.2.3. Transformation using DH5–alpha cells
Transformation was carried as instructed in Methods2.4.
The cloned VDR was placed in front of the Firefly and Renilla luciferases. A microplate was set up
for the cell transfection and the cells were transfected into HEK293T cells (Method2.5.). A dual
luciferase assay was then run and luciferase ... Show more content on Helpwriting.net ...
This WT stimulation was expected. The difference seen between WT1 and WT2 is due to the use of
two different clones being used. The mutants were found to not be acting as expected. However, the
mutant UGG is higher than the WT UGG. It is possible that is as a result of the mutant having not
been stimulated by calcitriol. Because of this, a fold stimulation plot was produced (Figure 6b). Also
with the UGG mutant codon, it can be seen that the calcitriol is not responsive when transcription
does not stop at the first stop codon.
A final Western Blot was done to analyse the transfected cells.
24 tubes were arranged, each receiving 2.5 μl from each well on the transfection plate to give a total
volume of 10 μl of each example because each transfection has four samples available.
Two separate polyacrylamide gels were run at 100V for 60 mins. The gels were transferred to a
nitrocellulose membrane and treated with primary and secondary antibodies (Methods2.6.). This
was followed with three 5 minute washes with PBS–T, and scanned. The results can be seen in
figure 7 as

Lactoglobulin Lab Report
Separation and Identification of Bovine Milk α–lactalbumin and β–lactoglobulin Corbin Croom,
Tyler Donathan, Cama Dooley, Nicholas Johnson October 15, 2014 Abstract Analytical gel
electrophoresis has been used to separate and identify many kinds of proteins including α–
lactalbumin and β–lactoglobulin. Both proteins can be derived from bovine milk. Skim milk was
centrifuged with a variance in pH to precipitate casein proteins. Whey material was filtered and
eluted through a Sephadex G–50 column. UV Spectrophotometric analysis confirmed the presence
of the protein within the fractions. Separation and identification of α–lactalbumin and β–
lactoglobulin was determined with SDS–polyacrylimide gel electrophoresis. With the molecular ...
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fraction number. These peaks occurred at fraction number 43 and fraction number 50, with
absorbances close to 1. Around these fraction numbers was where the largest elution of protein and
likely α–lactalbumin was present. Due to high amino acid content of phenylalanine, tryptophan, and
tyrosine, absorbance occurs at 280 nm. The high 280 nm absorbance would suggest a higher amount
of protein elution in the fraction. The early fraction numbers had significantly high absorbance
between fractions 4 and 9. This was due to quick mobility of the protein through the sephadex
column during the size exclusion chromatography before fractions were gathered. The absorbance of
the protein fragments: 7, 8, and 9 were all between 0.59 and 0.62. Protein fragments with
absorbance values above 0.5 had a noticeable amount of desirable protein within the fraction. The
electrophoresis gel was labeled in comparison with a pre–stained protein ladder as shown in Figure
2. The 10 kDa served as a base for the accuracy of the other protein ladder measurements. The 17
kDa measurement on the protein ladder was compared with the band values of the protein fractions.
α–lactalbumin has a molecular weight of 14.2 kDa. The second protein band that eluted from the
fractions in the gel was significantly close to 14 kDa in comparison with the 17 kDa measurement,
revealing that α–lactalbumin was present in the fractions. β–lactoglobulin

Sds Page (Sodium Dodecyl Sulfate Polyacrylamide Gel...
Gel electrophoresis is a routine laboratory procedure in biochemical studies that takes advantage of
a protein's amphoteric nature to determine its molecular weight and charge by running the sample
through a gel matrix under the influence of an electrically charged field. A popular example of gel
electrophoresis is Sodium dodecyl sulfate–polyacrylamide electrophoresis or SDS– PAGE which
has been used in this experiment to supposedly determine albumin and casein's molecular weights
respectively. The system used in gel electrophoresis consists of 3 major parts: a stationary phase, a
mobile phase) and an electrical power supply.
In SDS–PAGE the polyacrylamide gel serves as the stationary phase, the buffer (Tris–HCl in this
case) as the ... Show more content on Helpwriting.net ...
On the other hand, the Tris–HCl buffer and deionized water provides the system its shape since the
polyacrylamide complex is just a thin film of interlocking polymer fibers without any liquid to trap
within.
Polyacrylamide gels are usually preferred over other gel types such as agarose in protein
electrophoresis because of various benefits such as its ability to be cast in various pore sizes which
provides options for the researchers who observe protein samples with various Kda's depending on
how separated they aim the bands to be or how well the sample is sieved. It is also preferred because
of its charge neutrality upon setting as it does not interfere with the electrophoresis which is highly
reliant on the sample and the surrounding's charges within the system. Lastly, the gel itself is
colorless and protein stains do not adhere to the gel so there will be minimal problems with
unnecessary stains that aren't from the staining solution.
APS and TEMED was used to initiate the binding of acrylamide to bisacrylamide via a vinyl
addition reaction where TEMED catalyzes the decomposition of APS into free sulfide molecules
which then initiates the formation of the polyacrylamide complex.
Sodium dodecyl–sulfate or SDS is a protein denaturant that destroys weak interactions within the
protein molecule which unfolds it

Recombinant Dna Technology As An Environment For Separation
Recombinant DNA technology is genetic engineering process for forming a new gene. The gene
required is taken from the donor and joined with the carrier gene which is then inserted into the
vector. This method is used to create a vector containing gene from Bacteria sp Yp1 and
Esterobacter asburae YT1, which are then inserted to egg through microinjection. Microinjection is
a process where screw held syringe is loaded with required DNA or RNA and inserted to animal
cell. By this technique the cloned gene is inserted to egg of earthworm. The egg hatchs and develops
further producing transgenetic species having gene of gut bacteria.
Sodium dodecyl sulphate PolyAcrylamide Gel Electroporesis (SDS–PAGE) is a technique used for
separating protein based on their size and structure [14]. Sodium dodecyl sulphate (SDS) is an
anionic agent applied on proteins for linearizing them and to impart negative charge on the proteins.
When electric field is applied on protein covered with negative charge, they move towards positive
pole but no size separation can be seen. So PolyAcylamide gel is used as an environment for
separation. As electric potential is applied on proteins present in PAGE, it creates even distribution
of charge per unit mass resulting in fractionation of protein based on their size[15]. SDS–PAGE is
useful technique for acquiring the required protein. Once the transgenic Earthworm is created, this
technique can be used to acquire the specific protein/enzyme responsible for

Homoserine Synthesis Lab Report
The purpose of this experiment was to determine the cloning, transformation, DNA purification and
protein purification of enzyme homoserine transsuccinylase from Streptococcus pneunomiae
(SpHTS). The PCR result showed that there was no band in the DNA gel electrophoresis, which
means the experiment was not successful. The transformation process presented that there were no
colonies growing in the agar plate. The plasmid DNA had been purified and the concentration
measured was 25.2 ng/μL. The restriction digest sample had two visible fragments at 3000 and 3600
base pairs. There were five fractions of protein purification, which indicated five different protein
extraction. The protein gel electrophoresis showed that the flow through fragment ... Show more
content on Helpwriting.net ...
First, PCR technique was used to amplify the SpHTS gene. PCR is a molecular biology technique
for replicating DNA without using a living organism, such as E. coli or yeast (1). The PCR process
was carried in a thermal cycler which denatures the double DNA strands. Then, primers was
annealed to the single DNA strand. Then, 4 primers were used from a given DNA sequence in the
experiment. Primer 1 and 2 were used for PCR sample A with around 500 base pairs, and primer 3
and 4 were used for PCR sample B with around 300 base pairs. Ligation process was followed,
where sticky ends was created by cutting the plasmid and the PCR product with restriction enzymes,
then DNA ligase created a recombinant DNA (1). Then, it was followed by the transformation
process in which the recombinant plasmid DNA was incorporated into E.Coli. The host cells were
replicated and large quantities of plasmid DNA containing ampicillin resistance were generated. The
project was followed by purification of plasmid DNA using GenElute™ Miniprep Kit. The kit is
based on alkaline lysis of bacterial cell followed by adsorption of DNA into silica

You can view macromolecules by using a technique called gel electrophoresis. Gel electrophoresis is
a process used by scientists to study and view macromolecules. This process separates the different
macromolecules and coats the different macromolecules so you can easily see them. A
macromolecule is a very large molecule that contains many atoms. Agarose is used in gel
electrophoresis to coat the macromolecules. Agarose is made from agar or polyacrylamide. With gel
electrophoresis you can tell how many different things are put together to make one certain item
because this process separates all the other ingredients in the mixture. Also, the macromolecules
become coated in a negative charge during gel electrophoresis. The other side of the gel chamber is
positive and since opposites attract, the macromolecules are pulled to the other side of the gel
separating them. An electrical current is applied to the gel which causes the food coloring dye to
migrate and separate through the holes In the gel. Gel electrophoresis is also used in crime scenes
when identifying ... Show more content on Helpwriting.net ...
When I finish the experiment it will show each other color that was mixed together to make one
certain color. The other colors that were mixed together will show and each part will travel a
different distance to the other end of the gel chamber. I am using cut out combs to make holes in the
gel before I pour the gel. Electrodes will be placed at both ends of the gel chamber. I am also using a
buffer solution made of baking soda and bottled water. They will be hooked up to a positive and
negative clip which is hooked up to 5 9–volt batteries. This will make the end with the food coloring
negative, and the other side positively charged. The other food colors separate because they are
attracted to the positive end of the gel. I will check the gel every 10–15 minutes until a good
migration of the food coloring dye can be

Cell Cycle Analysis Paper
Cell cycle analysis. PC3 (8×104 cells/well) and LNCaP (3×105 cells/well) cells were seeded in 6–
well plates in 1.5 mL of complete growth media. Cells were treated with 2.5 μM of free sorafenib or
an equivalent sorafenib concentration of SMA–Sor, 3 μM of free nilotinib or an equivalent nilotinib
concentration of SMA–Nilo, DMSO or SMA for 48 h. Cell cycle distribution was assessed using
propidium iodide staining, as previously described {Somers–Edgar, 2011 #82}. Samples were
analysed using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the
proportion of cells in each of G0/G1–, S– and G2/M–phases were determined using CellQuest Pro
software (BD Biosciences, San Jose, CA, USA). Apoptosis analysis. PC3 (8×104 cells/well) ...
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The cells were washed with PBS and then incubated in serum–free media and treated with 2.5 μM
of free sorafenib or an equivalent sorafenib concentration of SMA–Sor, 3 μM of free nilotinib or an
equivalent nilotinib concentration of SMA–Nilo, DMSO or SMA for 48 h. Following treatment,
media was collected, centrifuged to remove cell debris, and freeze–dried for 12 h. Samples were
rehydrated and mixed with loading buffer (0.4 M Tris, pH 6.8, 5% SDS, 20% glycerol, 0.03%
bromophenol blue). For zymography, samples were loaded on a 10% SDS–polyacrylamide gel
containing 1 mg/mL of gelatin. After electrophoresis, the gels were incubated in renaturing solution
(2.5% Triton–X–100 (w/v)) for 30 min at room temperature and then for 24 h at 37°C in a
developing buffer containing 50 mM Tris, pH 7.5, 200 mM NaCl, 4 mM CaCl2, and 0.02% NP40.
The gels were then stained with Coomassie blue R250, and regions without staining were indicative
of gelatin lysis. The gels were briefly rinsed and scanned. For MMP–9 and isthmin–1 secretion,
samples were loaded on a 10% SDS–polyacrylamide gel, and the expression of MMP–9 and
isthmin–1 assessed by western blotting using anti–MMP–9 (D6O3H, Cell Signaling) and anti–
isminth–1 antibodies (Biorbyt, San Francisco, CA,

Structural Differences
1. What are the structural differences between GFP and BFP? Should these differences lead to
differences in when the proteins elute (exit the column)? GFP has 238 amino acid residues and has a
molecular weight of about 40,000 daltons. BFP is a derivative of GFP. It has a His–66 substitution at
the Tyr–66 position and a substitution from Tyr–145 to Phe–145. GFP is relatively unstable and
requires most of its amino acids to remain intact for its structure and fluorescence to remain as well.
These differences cause BFP to elute faster than GFP. 2. Why is it called chromatography? What is
the purpose? Chromatography is the process by which a chemical mixture is separated, so it can be
studied further. The objective to this lab was to separate

Gel Electrophoresis: Separating DNA and RNA
Gel electrophoresis is a procedure used in laboratories to separate DNA, as well as RNA and
proteins. A gel slab is placed in a buffer–filled box and an electrical field is applied. The negatively
charged DNA will migrate towards the positively charged side, where it can then be recorded and
further analyzed. An example for the use of gel electrophoresis would be in identifying people. DNA
is present in almost every cell of our body. Each person has a unique sequence of DNA base pairs
that makes up our DNA fingerprint. A DNA fingerprint is the same for every cell, tissue and organ
of a person. According to Dalya Rosner on the Naked Scientists website, "DNA fingerprinting is a
technique for determining the likelihood that genetic ... Show more content on Helpwriting.net ...
The more agarose that is used and dissolved, the firmer the gel will be. Typical concentrations used
are between 0.3% to 2% (Buckingham, 2012). The concentration depends on the type of analysis
needed. A higher concentration of agarose, making a stiffer gel, would be used to detect smaller
DNA fragments (100–3000 bp) while a lower concentration would be used for larger fragments
(5000–60,000 bp). For pieces 50,000 bp and over, pulsed field electrophoresis is used where an
alternating current is applied (Buckingham, 2012). A gel comb is placed into one side of the gel
mold to create holes (wells) in the gel. The gel will need to cool and solidify, about an hour. The
comb is then removed, which leaves the empty wells in the gel. Buffer is poured into the
electrophoresis box, usually a horizontal acrylic container. The gel, still in the mold, is placed into
the buffer inside the box. The gel is slightly submerged in the buffer which will conduct the
electrical current in the gel. With a micropipette and a new pipet tip, a loading buffer, which
increases the density of the sample, is added to the DNA sample in a tube. The DNA sample is
colorless, so a tracking dye, such as bromophenol blue or xylene cyanol is used to visually track the
DNA movement. The dye migrates at a specific speed similar to the DNA. The sample is then
transferred into the first well in the gel. With a new clean

Analysis Of Two Dimensional Electrophoresis ( Ief )
TWO–DIMENSIONAL ELECTROPHORESIS (IEF)
The different methods of separations of a mixture, to get one particular constitute of the mixture are
available now. The separation methods are based on the charge present and on migration rate and on
applied electric field are known as electro–kinetics methods. Many methods are available which are
based on the electro kinetics method. Methods like electrophoresis, isotachophoresis, isoelectric
focusing and related techniques are available for the separation the components from the mixture.
This separation of molecules depend upon the many parameters like temperature, pH, ionic strength,
viscosity, applied electric field, concentration of electrolyte, surface charge, net charge of molecule
etc. These parameters are very important for separation.
Electrophoresis is the technique which monitors the charge of the molecule under the electric field
applied and separates the molecule by considering parameters like electrophoretic mobility, charge,
size of the molecule.
Isoelectric focusing requires constant electric charge with the different pH gradient on the gel and
due to this molecules get separated according to their isoelectric point.
Isotachophoresis uses different electric field with the combination of pH gradient for the separation
of the molecules or charged species.
 Electrophoresis:
The basic principle of electrophoresis involves the separation and isolation of charged molecules
due to their differential migration in a buffer

Macromolecules Using Sds
The testing of various proteins was performed by comparing the molecular weight of proteins using
SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules
consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy
of structure that consists of folding that determines the direct function of each protein.. The
molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium
dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the
protein structures and causes them to separate due to the change in bonding charge. SDS and heat
are how the proteins are denatured. The process of denaturing a protein is breaking

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

Sds Lab Report
SDS page
Name: Shelby Clark
Lab Partners: Kaliah Goodman, Howsikan Kugathasan, and Suntesia Bowen
Date of Laboratory: September 21, 2016
Goal of Laboratory:
The goal of this laboratory was to successfully make a gel and to run an SDS–PAGE and determine
molecular weight (MW) of an unknown protein sample by comparing it to Log10 of the migration
of molecular weight of the standards.
Background:
The purpose of SDS–PAGE is to separate proteins according to their size, and no other physical
feature. SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules
but also has a negative charge (sulfate) attached to it. Therefore, if a cell is incubated with SDS, the
membranes will be dissolved, all the proteins will ... Show more content on Helpwriting.net ...
Therefore, if we had many copies of two different proteins that were both 298 amino acids long,
they would travel together through the gel in a mixed band. As a result, we would not be able to use
SDS–PAGE to separate these two proteins of the same molecular weight from each other. Some
possible errors that occurred during this lab include poorly executing inserting the MW marker
solution into the well of the gel. This caused not so clear bands as shown in figure 1. Incorrect
measurements of the migration could also lead to errors in the data. The molecular weight of the
protein was determined by plotting a graph of the Log MW vs migration (cm) and generating a
standard curve. The unknown sample molecular weight was calculated as 79.43 kD. Experimental
errors in this lab could be minimized by carefully loading the wells on the

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