The Isolation Of Milk Whey

The Isolation Of Milk Whey
Day 1: Isolation of Whey The isolation of milk whey began with 10 mL of nonfat milk which had been centrifuged at 16,000 x g for 45 minutes in
a refrigerated centrifuge. The top layer***. 10 mL of the nonfat milk were then pipetted into a small glass beaker. The pH of the nonfat milk was
slowly adjusted from a pH of 6.60 to a pH of 4.60 through the dropwise addition of 0.5 M and 0.05 M HCl. The coagulated solution was heated to
approximately 40В°C for 30 minutes while being constantly stirred.
While the solution was being heated, 100 mL of elution buffer was made using 0.02 M Tris, 0.5 M NaCl, and 0.02 M imidazole with a final pH of 7.0.
100 mL of binding buffer and 100 mL of stripping buffer were obtained as well. The binding buffer had... Show more content on Helpwriting.net ...
The previous wash and collection of fractions was done again using the elution buffer. All 10 fractions collected were analyzed using UV spectra at
280 nm using the *** as the blank.
Day 3: Protein Characterization Using SDS–PAGEGel Electrophoresis and BCA Assay A crude whey sample and a purified *
–lactalbumin sample
were prepared for SDS–PAGE gel electrophoresis by mixing 20 ВµL of each sample with ВµL of the reducing gel buffer in Eppendorf tubes. These
two samples were then boiled for 5 minutes and then allowed to cool to room temperature. A precast gel was inserted into the gel running
apparatus and the comb was removed. Consequently, a 10x stock solution of tank buffer was diluted tenfold and added to middle of the chamber
until it covered the gel. Then, * ВµL of crude whey sample was added to well 10 and the purified sample was pipetted into well 4. A standard MW
ladder was loaded into well 1 of the gel. The remaining buffer was poured into the bottom the tank. The top was placed and the gel was then run at
a constant 150 V for ***. After running the gel, it was removed from the apparatus and was rinsed several times with water. To fix the gel, it was
rocked in a container with 50 mL of water and 1 mL of glutaraldehyde for 10 minutes. The glutaraldehyde mixture was decanted and enough gel code
stain to cover the gel was added to the container. The container was
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Lab 1 Carbohydrates Bch2333 Essay example
Lab BCH 2333
Section:
Lab 1 Carbohydrates: Separation Techniques Based on Molecular Size
TA:
Wednesday, January 16th, 2013
Team #4
By:
Partner:
Purpose
The purpose of this experiment is to exemplify how differences in molecular weight allow separation of polymers from their monomers. Methods of
dialysis and gel filtration chromatography will be used to separate a glucose monomer from a starch polymer. Colorimetric glucose oxidase assay will
be used to monitor the presence of glucose and a colorimetric iodine assay will be used to monitor the presence of starch in prepared solutions after
separation
Results and Discussion
Table 1: Glucose oxidase ... Show more content on Helpwriting.net ...
R6

[pic]
**Glucose and Starch graphs are separated because of the significantly different concentrations of the two which obscures the graphs when plotted
together
Figure 1 Concentration of glucose relative to elution volume. Graph plotted using Excel. The equation of the line is represented by a 6th order
polynomial (y = –4E–08x6 + 8E–06x5 – 0.0006x4 + 0.0184x3 – 0.2952x2 + 2.0705x – 4.6828) with a regression RВІ = 0.75191.
[pic]
Figure 2 Concentration starch relative to elution volume. Graph plotted using Excel. The equation of the line is represented by a 6th order polynomial
(y = 1E–09x6 – 2E–07x5 + 1E–05x4 – 0.0004x3 + 0.0063x2 – 0.0379x + 0.0784) with a regression of RВІ = 0.4605.
Analysis of Graphs
Three peaks are observed in Figure 1 (concentration of glucose vs. elution volume) which was expected due to the results in table 4 that show
intervals of elution. The intervals of the elution are represented as peaks on the graph. The intervals are due to the glucose molecules that enter the
beads of the column causing the glucose molecules to elute slowly. Two peaks are observed in Figure 2 (concentration of starch vs. elution volume),
which was not expected. One peak was expected for the

Rgfp Synthesis Lab Report
Results
The induction process for the activation of rGFP was vital to this experiment, to ultimately determine the presence and molecular weight of rGFP from
crude extract. T7 RNA Polymerase activates the T7 Promoter, by binding to it upstream of the rGFP gene, in the presence of IPTG, allowing the
expression of rGFP. The expression results from the transcription of the rGFPgene that produces rGFP mRNA, which is then translated to produce the
rGFP. By inducing the IPTG activation of theDNA regulatory sequence T7 Promoter, as seen in Figure 1, the presence of rGFP became apparent under
the UV lights after the Ni+2 Agarose Affinity Chromatography washes and elutions. The G3 sample, three hours post–induction of the IPTG sample,
had the highest fluorescence at 14901 RFUs. ... Show more content on Helpwriting.net ...
This purified form of rGFP was produced by manipulating the lysing effects of the freeze/thaw cycle. Since this cycle was not 100% effective, the
pelleted debris fluoresced as it was the presence of some rGFP protein that causes activity, not purity. The W1 didn't have any hints of fluorescence
because only the void volume passed through the Ni+2 Agarose Column. However, the W2–W4 had slight fluorescence because some rGFP was
washed into them, as the column was overflowed, with only a specific number of binding sites. The E2 fraction had the highest fluorescence activity,
based on the microplate reading of 2621.9

Analgesic Drugs Lab Report
TLC Analysis of Analgesic Drugs Author: Monique Amanda Mendez Lab Instructor: Wenmo Sun Organic Chemistry Lab 243A: Section 038 Date
Work Performed: 02 September 2015 Discussion: Lab 1a– TLC Analysis of Analgesic Drugs Discuss the relative polarities of the components of the
analgesic drugs based on their functional groups The relative polarity of the analgesic drugs depends on their functional groups. Polarity of the drugs
depends on several differentiating factors which include how the compound can hydrogen bond to itself or another compound, the number of
electronegative atoms that are present within the structure of the compound, the net dipole moment of the molecule and the polarizability of the bonds
or atoms that are present within the compound, which can highly define how polarizable the compound is or will be. The more polar the functional
group, the stronger the bond is to the stationary phase, making it slower for the molecules to move down the TLC plate, thus the stronger it will be
absorbed on the surface of the solid phase.... Show more content on Helpwriting.net ...
A compound's structure will affect its polarity, along with affecting the Rf values due to the polarity of the compound. The least polar the compound
the farthest it'll move on the TLC plate, the more polar the compound the less it'll move on the TLC plate. The two compounds that I used in lab
consisted of Caffeine and Ibuprofen. Caffeine is a very polar molecule, due to the carbonyl groups that are present within the compound, these two
functional groups of carbonyl on the Caffeine structure greatly add to the polarity of the compound, along with the lone pairs of the nitrogen that are
present also affect the polarity of the compound. Ibuprofen is significantly less polar than caffeine, but had a much greater Rf value than caffeine due
to the polarity (since it is less polar than

Dneasy Extraction Kit
One of the primary goals of this experiment is to generate a genomic library for A. fischeri, and the first step to do this involves isolating and
analyzing the chDNA of the bacteria. This was completed using the Qiagen DNeasy Extraction Kit, which involves a variety of steps including: lysis
of the chDNA, the binding of the sample to silica, multiple washings of the sample, and an elution to obtain pure chDNA for analysis (Qiagen, 2006).
To start off, 180 Ојl's of resuspension buffer was added to the A. fischeri pellet in a 1.5 ml microcentrifuge tube (Qiagen, 2006). In order to deactivate
nucleases (so that nucleic acids can be released), 20 Ојl of proteinase K was added (Qiagen, 2006). After vortexing the sample in order to mix
everything... Show more content on Helpwriting.net ...
Special care was taken to ensure that the spin column did not come in contact with the flow–through. Since the chDNA is bound to the silica at this
point, they must be separated using two wash buffers. The initial 500 Ојl of buffer (AW1: Wash Buffer 1) was added in order to remove contaminants
and proteins that could be in the sample using chaotropic salts (Qiagen, 2006). After centrifuging the sample for 60 seconds at maximum speed, 500
Ојl of Wash Buffer 2 (AW2) was added to remove the salts (this contained ethanol). After centrifuging the mixture for 3 minutes at maximum speed,
AW2 was added again and centrifuged (Qiagen, 2006). Once this final centrifuge was finished, the sample was air–dried for 10 minutes at room
temperature. Next, a microcentrifuge tube was added beneath the spin column, and 75 Ојl of DNA grade water was added to the spin column. This was
done to rehydrate the DNA and separate it from the silica (Qiagen, 2006). The tube was then incubated for 2 minutes at room temperature and
centrifuged at maximum speed for 2 minutes. This step was repeated in order to elute the sample (total volume 150 Ојl).
Once the elution had been completed, the sample obtained was further analyzed using a Nanodrop 2000 Spectrophotometer. The purity and
concentration of the

Expression and Purification of Recombinant Linker Histone...
Two restriction enzymes, Nde1 and Xho1, enabled the gene coding for the H1a21c protein to be successfully inserted into the pET22b plasmid.
However, since the pET22b vector contains a His– tag sequence, it was important that the Xho1 enzyme to introduce a stop codon that prevented a
His– tag to be attached to the H1 protein.
The SP FF 'fast S' column was used to purify the H1 protein based on the ionisation state of the protein. Referring to the structure of H1 proteins, they
contain a high level of lysine residues (compared to core histones), thus making them relatively basic (Johns, 1971; Cole, 1984). The cysteine residue
increased this positive nature of the H1 protein, allowing the isolation of this protein using a cation exchange column.
The strong anion ligand, Sulphopropyl, allowed the protein to stick to it via ionic interactions during the low salt concentrated elution process. Most
unbound proteins eluted, however, proteins containing positive charges eluted simultaneously with the H1a21c protein during the high salt
concentrated elution process. Gradient elution (increasing salt concentration) was used and this is seen by an increase in conductance as the proteins
eluted (figure 3). A consistent increase in conductance confirmed that the elution process was correct.
The H1 proteins, as shown in figure 3, eluted in the last peak which showed a low absorbance due to a lack of reactivity. The eluted H1 protein was
applied to an SDS gel in order to confirm the

Csa Concentration Varied Between Rounds ( Table 1 )
CsA concentration varied between rounds (Table 1). Library was ordered from Integrated DNA Technologies (IDT). N30 library (30 unknown
nucleotide bases): 5 '–GGA GGC TCT CGG GAC GAC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GTC GTC CCG ATG CTG CAA
TCG TAA–3 '. Capture Sequence: GTC GTC CCG AGA GCC ATA/3BioTEG/. Primer_P1: GGAGGCTCTCGGGACGAC. Primer_P2:
TTACGATTGCAGCATCGGGACG. P2_biotin: /5BiosG/TTACGATTGCAGCATCGGGACG. All oligo's (primers, library, and capture
–strand) were
ordered from IDT . Bio–capture used: 5' biotin from IDT. Two SELEX buffers were used: SELEX 1 buffer (10 mM HEPES, 150mM NaCl, 5mM KCl,
and 2mM MgCl2) and SELEX 2 buffer (10mM HEPES, 150mM NaCl, 5mM KCl). The composition of the PCR mix was as follows: 10% 10X buffer,
.5% Taq, 1% P1, 1% P2, 2% dTNP, and 80.5% water. All gels were run in .5X TBE buffer and 3% agarose gel. Columns used: Micro Bio
–Spinв„ў
Chromatography Columns. PCR polymerase used was Choice Taq from Denville Scientific. DNTP is also from Denville Scientific. Streptavidin Beads
are from Sigma Aldrich.
4.2 The SELEX Process The general steps of the SELEX process are; the binding reaction between the aptamers and the target, washing steps to remove
the unbound DNA, PCR amplification of the bound DNA, and strand separation of the DNA back into ssDNA [3]. The Capture–SELEX cycle consists
of three main sections: Elution profile, Library Amplification, and Strand Separation. The elution profile section includes the adding of biotin to the
library

Gel Filtration Chromatography Lab Report
Gel–Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different
biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to
allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the
column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column
first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will
have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to
the molecules size.
Methods:
Day one ... Show more content on Helpwriting.net ...
Then measured the absorbance of blue fractions that are within 5% of before and after the absorbance of the darkest blue one. Repeated the same steps
with the yellow fractions, measured them at 440nm.
V_o = void volume, which is calculated by locating the peak absorbance at 650nm the darkest blue one, total the volume up to and including the peak
absorbance fraction. V_t = total volume of the column, where the fraction of the peak absorbance at 440nm the darkest yellow, total the volume up to
and including the peak absorbance fraction.
Day Two Elution of myoglobin:
A horse heart Myoglobin sample was loaded into the gel column, the same way it was loaded on day one. Fractions were collected at 1.0 mL in each
Eppendorf tube, and weighed after collected. Collected fractions until the red had run clear out of the column.
Determining the absorbance of myoglobinгЂ–

Creatic Pollution : The Nature Of An Isocratic Elution
The column volume was calculated to be 18 mL. The volume of the resin stationary phase was measured to be 15ml. Based on figure 1 above, the
void volume is approximately 3 mL. the first three points on the graph have little to no absorbances at the wavelength of absorbance for the compounds
of interest. This indicates that those first three collections only contain void volume. The next (fourth) mL collected as seen in figure 1 has not reached
the max absorbance and therefore also contains some void volume. So, blue dextran, the compound contained in the 4th mL collected eluted in the void
volume. Consequently, the void volume is at least 3 mL however, the exact volume cannot be determined.
5.An isocratic elution is accomplished when the mobile phase used remains constant throughout the separation. A gradient elution is when the nature of
the mobile phase is increased at a continuous rate during the separation. Finally, a stepwise elution is when the concentration or composition of the
mobile phase is changed during the separation in a gradual manner (Iverson, 2017). Isocratic elution is useful when one has a mixture of compounds
with large differences in their chemical or physical properties. For example, the compounds have a large difference in their molecular weight and so
size exclusion chromatography with an isocratic elution is performed. Gradient elution is effective with components of a mixture with a wide variety of
polarities. Gradient elution is useful in ion

DNA And Crime Scene Analysis
Each year about 33,000 crimes are solved by DNA database, therefore, showing that DNA has an important element when determining and finding
potential suspects who may have committed the crime. DNA, or deoxyribonucleic acid, is a substance that makes up an individual's genetic identity.
DNA is found in all organisms, but more the abundance of DNA is found in cells which include a nucleus or "nucleated cells"("preservation and
collection of biological evidence"). Within a crime scene detectives and officers can use common materials such as tissues, pillows, a bottle, and
more. When a detective finds cases in which DNA is present, they can take the samples back to lab to extract and quantify the DNA to see if they can
do further analysis on ... Show more content on Helpwriting.net ...
However, since hair is typically made up of the protein, Keratin, it is typically harder to extract DNA from hair. In order to extract DNA from hair it
is important to use the follicle of the hair. DNA is easily found in the root and follicle of the hair because it is connected to the blood stream
(Heywood, 21–22). When attempting to extract the DNA from the hair, forensics attempt to wash out all of the protein, therefore, the only thing left
is the pure DNA sample. However, there is not a 100% chance that protein will be washed out completely, meaning that there is a chance that DNA
could be contaminated, in other words the DNA would be hard to read and find a yield. In addition, due to this fact the average success rate of yielding
DNA from hair is 60–70% ( "A Simple Method of Genomic DNA Extraction from Human Samples for PCR–RFLP Analysis"). However, unlike hair
cells, cheek cells are much simpler to extract DNA from. This is due to the fact that the tissue inside of your mouth, contains a lot of available DNA
when the area is shedded (Smith, 43). When the popsicle stick is rubbed along the tissue it allows for cells to be shedded, therefore it is easy to obtain
the cells, when one rinses saline buffer inside of the mouth and spits back into a microcentrifuge tube (Smith,

Drosophila Melanogaster Lab Report
Introduction Genes are unique segments of DNA and in this laboratory experiment, the Actin gene of Drosophila melanogaster will be extracted and
amplified with various laboratory processes including PCR, ligation, and transformation. Also, the gene that was extracted will be confirmed and
sequenced with the process of cycle sequencing and with the help of NCBI database. The DNA that is first extracted will be referred to as "genomic
DNA" because it was extracted directly from the fruit fly, but later on, it will be referred to as "plasmid DNA" and this is when it is incorporated in the
plasmid. Drosophila melanogaster or the fruit fly is one of the most commonly used organisms in genetic experiment. They are commonly used
because of their small size, four homologous pair of chromosomes, easy maintenance, and easily observable traits (Pierce, 2012).
Groups extracted either the 18S or the Actin gene of Drosophila melanogaster. The 18S gene is located on the flies X chromosome and has a length of
488 base pairs. The Actin gene is located on chromosome 3 of the fly and it has a length of 4,760 base pairs (Adams, 2000), but we are ... Show more
content on Helpwriting.net ...
In this case, our plasmid DNA is isolated from a liquid culture of the E.coli that was transformed. This is done by reacting the liquid culture with five
buffers. The buffers are P1, P2, N3, PE, and an elution buffer. P1 is used to re–suspend the pellet and degrade RNA, P2 is used to lyse cell membrane,
PE is used to wash the sample, the elution buffer is used to release DNA from the spin column, and N3 is used to precipitate proteins and genomic
DNA. The main components of P1 are Tris, EDTA, and RNase. The main components of P2 are NaOH and SDS. The main component of PE is ethanol
and the main component of N3 is acetic acid. The main component elution buffer is water. The possible contaminations of mini–prep are proteins and
salts (Garey et al.,

Separation Of Fluorene And 9 Fluorenone
In this experiment, separation of fluorene and 9–fluorenone took place using column chromatography. The pureness of the separate compounds was
checked using TLC and melting point analysis. Rf values were the determined from the TLC plates and calculated to compare the experimental Rf
values to the starting mixture Rf values. TLC is useful to separate tiny samples and is considerably quick, while chromatography is used to separate
considerably larger mixtures but takes more time. The melting point of both desired molecules was found and contrasted with the actual values. The
melting point is used to assess the purity of each compound recovered. Column chromatograpahy separates compounds based on polarity. Column
chromatography contains a mixture dissolved in solvent and put in a column that had solid adsorbent and an eluent. There are different phases in the
column: a polar stationary phase (solid adsorbent), the alumina, and mobile phases (eluent), which can flow throughout the column. There are different
phases due to different polarities, resulting in dissimilar bands. TLC separates compounds due to polarity. Each sample is dotted on the plate and
placed in eluent and the samples quickly separate due to polarities and adsorption. The spots can either move far or short distances. The weakly
adsorbed compounds move faster than the stronger adsorbed components. The textbook mentions simple elution stepwise elution to elute each fraction
in the column chromatography. Simple

Thin-Layer Chromatography Lab
Introduction Thin–layer chromatography, also known as TLC, is a principle that describes how various compounds travel multiple distances when
placed as a thin layer on a plate. TLC is a technique that can be used to determine how many components are in a mixture. TLC can also be used to
determine a specific compound in a mixture. After performing TLC, the retention factor (Rf) can be used to determine a specific compound in a
mixture. The retention factor (Rf) is During TLC, there is a step called elution. In elution the components on the TLC plate will have different
solubilities and different adsorption strengths. The liquid at the bottom of the TLC chamber is called eluent. The purpose of this experiment was to use
TLC to separate and identify... Show more content on Helpwriting.net ...
If the solutions are placed to close together on the TLC plates, then is possible that after being place in the TLC chamber, they will end up close
together and it will be difficult to distinguish which spot belongs to a certain solution. Another way to improve this experiment would be to make
sure that the bottom of the TLC plate is parallel to the bottom of the jar in the TLC chamber. If it is not parallel, then the solvent front will not
develop properly and it will make determining the spots or comparing the spots more difficult. The last way to improve this experiment would be to
make sure that the .5% glacial acetic acid in ethyl acetate inside of each chambers does not evaporate. If it does evaporate this could affect the
development of the solvent

Claisen Rearrangement
The Claisen rearrangement is a [3,3] sigma tropic rearrangement. This is a class of reactions that is pericyclic. The stereochemistry is determined by
the transition state (generally chair–like). Out of keto and enol, the more stable tautomer is enol. Enol is a more stable tautomer because the benzene
loses aromaticity when it is in its keto form. The IR data of spectrum 1 supports this answer. The spectrum shows the OH bond, which shows an enol
form. Usually, the keto form is more favorable than the enol form, but because re–aromatization is more stable, the enol form is the more stable
tautomer. The peak at 3.37 is significant of the starting material. The peak at 4.17 is significant of the product. The peak at 2.55 is significant of phenol.
... Show more content on Helpwriting.net ...
Plugging in 120 minutes for 2 hours of reflux, the maximum yield of product that could have been achieved in the given amount of time that the
reaction was at reflux would be 63.489% multiplied by the moles of starting material (3 mL allyl phenol ether gives 0.022 moles). This gives
0.0138 moles of product as the highest percent yield possible. The mass of the product recovered was 0.5843 grams. This mass represented only a
19.9% yield. The mass is way below the potential percent yield at the end of a 2.00–hour reflux compared to the GC/FID data. At 2 hours and 15
minutes, the potential percent product yield was about 70.9%. This is much greater than the result after 2 hours (if not a little less) of reflux during
the lab period. The actual yield at the end of the lab session at 19.9% is ~ 50% lacking from the posted data rate of 70.9%. If more time were
provided for reflux, more of the product would form. The Claisen reaction needs a lot of time to reflux, much more than (up to) 2 hours that were
provided during the lab

DNA after synthesis were PCR amplified using pFx enzyme...
DNA after synthesis were PCR amplified using pFx enzyme respectively for both premature AI and mature AI. The products of the PCR have blunt
ends due to the amplification done by pFx. Mature AI was not able to be amplified using pFx so it was done using Taq Polymerase. PCR clean up of
the products were done and was quantified using Nanodrop. Restriction digestion of the Mature AI, Premature AI and pET28–a was done at the Xho
and Not1 restriction sites. Purification of the digested products were done using Sodium acetate precipitation protocol. Ligation of the purified
products were done using Ligase enzyme. Stage 1Stage 2 (Cycle 33)Stage 3 95вЃ° C95вЃ° C55вЃ°C68вЃ°C68вЃ°C4вЃ°C 2:0 min
30Sec45Sec1:30min7:0 minв€
ћ Table.... Show more content on Helpwriting.net ...
Pellet was resuspended in buffer and then SDS–PAGE was run for the samples 3.5.5. ProBond purification of Premature AI and Mature AI: Lysis and
Suspension buffer composition for BL21 E.Coli cells protein expression are: Lysis buffer(pH 7.0): 50mM MOPSO, 10% Glycerol, 10mM MgCl2
Wash buffer(pH 7.0): Add 20mM Imidazole to the lysis buffer. Elution buffer(pH 7.0): Add 250 mM Imidazole to the lysis buffer. To remove
precipitate containing salts column was run (affinity chromatography).In affinity chromatography 1 ml raisin was taken which contained nickel ions.
It was equilibrated against tris buffer of pH 8. The equilibrated raisin was added in protein suspension then it was kept for binding for 2 hours. After
the binding process, the column was run i.e. affinity chromatography was performed. Histidine tags were present in the protein, it has affinity for
nickel ion, and so all Histags got binded with nickel. The elution fractions were collected which contained purified protein and its activity was checked
and SDS–Page was run. Before adding resin sample was taken out for assay. After bound sample was taken out in separate tube for assay. About 37
mL of the wash buffer was used to wash the undesired proteins. First two and the last two washes were saved for the activity. About 4 mL of the
elution buffer was used for eluting the desired protein. All fractions were

Purification of lactate Dehydrogenase
Purification of Lactate Dehydrogenase
Results and Discussion
Monitoring Assays Enzyme activity assay and Bradford dye binding protein assay was used.
Ammonium Sulfate Fractionation In order to isolate and purify Lactate Dehydrogenase (LDH), we first extracted LDH from bovine muscle tissue. We
minced 8 grams of meat and homogenized using blender. The homogenate was centrifuged and the pellet consisting of membranes, organelles,
cytoskeletal components and structural fragments was discarded. The crude extract was subjected to 40% (NH4)2SO4 fractionation. The purpose of
this is to remove molecules of lesser solubility than LDH like lipids, fats and low soluble proteins. For this, 9.24 g of was slowly added to crude
extract while stirring reaching final solution saturation of 40%. The mixture was then centrifuged and the pellet consisting the contaminants was
discarded. From table I, the data indicate that the yield for this was 88% with purification factor of 1.5. The total protein in 40% supernatant was 121
mg which decreased from 210 mg present in crude extract while retaining 88% of LDH suggesting some contaminant proteins were removed. Due to
some discrepancies in protein data for crude extract and 40% supernatant, we re–assayed these. Even though week 3 data may not be very reliable, the
crude extract assay from week 2 and 40% supernatant assay from week 3 gave the most appropriate data with least error. The 40% supernatant was
then subjected to 60%

Synthesis Of Lycopene
Conclusion The isolation of ОІ–carotene from lycopene via column chromatography was accomplished, but results were inconsistent. Many students
were unable to isolate the first elution compound, ОІ–carotene; however, lycopene isolation was very obtainable; due to the structural similarity of
these two molecules, it was intuitive that a separation would be difficult. UV/VIS and TLC analysis of the compounds confirmed that it is possible to
isolate ОІ–carotene from lycopene, due to differences in dipole moments of these chemicals. In future experiments, it is suggested to use a mobile
phase less polar than 15% ethyl acetate and 85% hexanes such as 5% ethyl acetate and 95% hexanes, for this would have potential to create a better
separation. It was

Synthetic Proteins And Cellular Components Essay
Now with the GFP in the lysate solution with other soluble proteins and cellular components, we need a strong and specific process that targets only
the GFP and not the other parts of the mixture. This strong and specific process used is called affinity chromatography, in which one substance can be
purified by using its specific interaction with another substance. In this case, GFP has six histidines at its N–terminus which can easily bond to
Ni2+and this interaction can be used to purify the GFP from the lysate by using a Ni–NTA column. This Ni–NTA column allows for solutions to
follow through except components that can easily bond to the immobilized Ni2+ ion in the column's silica matrix. Before we could use the Ni–NTA
column, we had to equilibrate it by adding some binding buffer to the column that washes and prepares the column and then centrifuge. After the
column is ready for use, the bacterial lysate can be added to the column, with some left for later analysis. The column is then centrifuged in order for
the mixture to flow through the matrix and separate those components that can bind to Ni2+ (not necessarily only GFP) and those that simply flowed
through. The liquid from the collection tube is collected and transferred into a tube and labeled "F" for flow through (Figure 1, lane F). At this point
the GFP is bound to Ni2+ in the matrix of the Ni–NTA column, however it is not alone. Many other proteins and cellular components may have bound
to the column as well due

Lab Report Essay
Discussion:
For each half of the membrane, only 1 unique band should appear in each sample lane, and the band should be consistent with other lanes as well. The
reason is that a primary antibody can only bind to a specific protein. If that specific protein is present in the sample, then a band should appear. In this
experiment, goat anti–rabbit HRP was used to localize the site of the primary antibody. This secondary antibody was used for both halves of the
membrane, because both primary antibodies were made from rabbit.
In theory, a LDH band should be observed in each of the sample 1~4, because all four samples contain LDH enzyme. A band was observed for each
well 1,2 and 4, which suggests that LDH enzymes are present. Sample 4's band however, is very faint. This suggests that the protein amount in the
loading is very scarce, resulting a weak band expression. Sample 3 is the same crude protein as sample 4, but it has 10–fold less protein amount for
loading than that of sample 4; therefore, well 3 would less than likely to have a band expression. The LDH bands appeared around 35 kDa according
to the molecular marker; however, the literature molecular weight for LDH is 140 kDa. The reason LDH bands ... Show more content on
Helpwriting.net ...
The band expressions suggest that BSA protein is presented in the sample. A band shouldn't have been observed in well 8, because it is a purified
LDH sample, which shouldn't contain any BSA protein. Since the band is very faint, the band appearance could be due to sample contamination.
The sample from well 7 or 9 could have overflowed to well 8, resulting a faint band in well 8. BSA protein has a molecular weight of 66.5 kDa,
which is quite close to what is observed on the membrane. Some other bands could also be observed in well 10. Those bands could just be the stains
that weren't washed away, or could have been incomplete separation of BSA protein due to electrophoresis or transferring

Lab Report Green Fluorescent Protein
Abstract section
Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this
experiments is to purify and express a His2–tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA–GFPUV > through
a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuvgene (UV–optimized GFP) was over expressed in the E.
Coil strain BL21 (DE3) (pLysS) as an n–terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain
purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay
was performed to obtain the total protein amount. When calculating the ... Show more content on Helpwriting.net ...
Below is the map of pRESTA–GFPuv in Figure 1. This map shows us that IPTG was induced first in the system, making the lac repressor inhibited,
this then expressed DNA T7 polymerase. This makes the T7 promoter to create the protein of rGFP is transcription. G0 means time equals zero.
FIGURE 1: An expression plasmid map. rGFP contains a total of 289 amino acids from the GFPuv DNA sequence shown in Figure 2. There is 238
amino acids in the rGFP and 38 amino acids in His6tag/Xpress epitope. His 6 tag comes from the N terminus.
FIGURE 3: A schematic diagram of the rGFP protein.
In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest
elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5
RFUs/mg. Overall, in the Figure 3, the E1–E6 had most of the rGFPs due to being bonded in Ni+2. W1–W6 rGFP is much smaller than the E1–E6
amounts.
FIGURE 3: The combined elution

Why The Enzyme Is Understanding Their Properties Essay
An important aspect of studying proteins is understanding their properties. The hypothesis of this experiment aimed to test whether the enzyme in
question, N–acetyl–ОІ–D–Hexosaminidase, has an overall negative charge and will elute from the DEAE column in the first protein peak. The
experiment was done in two parts. The absorbance values obtained from conducting the Lowry protein assay were used to extrapolate the total amount
of protein and calculate the concentration of the protein. The total amount of protein in the sample was 120Вµg and the concentration of the protein
was 24.0 Вµg/Вµl, which was within the allowed range of error. Anenzyme assay with paranitrophenol was conducted to determine the specific
activity of the enzyme. The specific activity of the isozyme HEX B was calculated to be 0.00009 units/mg and the specific activity of the isozyme
HEX A was 0.00004 units/mg. At the conclusion of this experiment, the data obtained from theion–exchange chromatography indicated that the
unknown protein sample contained two isozymes of the enzyme N–acetyl–ОІ–D–Hexosaminidase. It was observed that isozyme HEX A had an overall
negative charge and therefore eluted last from the DEAE column, whereas HEX B had an overall positive charge and did not bind to the column, hence
eluted first.
INTRODUCTION Ion–exchange chromatography is a separation technique that separates molecules and ions based on their overall net charge (1,6). It
can be used to separate amino acids, proteins, and

Fruit Fly Lab Report
In this experiment, there are many techniques to amplify and clone a gene from the fruit fly, Drosophila. The gene will be a homolog of a human
gene that is important for this research. Ten homogenize files and KAc/LiCl working solution was used for the genomic DNA extraction. The
buffer that was used contained ddH2O, Tris/Hcl pH7.6, EDTA, NaCl, and SDS. Cells are broken down so that the solution can release DNA.
Moreover, as more cells are broken down the more DNA will be isolated. Then, mixed the KAc/LiCl working solution thoroughly and waited for 10
minutes. Separation of cell debris from the nucleic acid occurred during this part of the procedure. Added EtOH so that the pellet will move through the
EtOH and recovered the pellet. Then, resuspend... Show more content on Helpwriting.net ...
The Multicore Buffer was used since it works well for most restriction enzymes. Added Bovine Serum Albumin (BSA) as a blocking reagent
because it sticks to the surface keeping more of the enzyme in the reaction solution. Digested 3 samples of plasmid DNA with each of the enzymes
from the 2 single digests and 1 double digest. Did three master mix reactions one for EcoR1, NcoI, and EcoR1 and NcoI (E + N). The resulted
enzyme volume is 5% or less of the overall reaction because it prevented star activity in which the enzymes could non–specifically cut DNA. Then,
incubate it at 37ЛљC for 20

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

Lab Report On Affinity Chromatography
Affinity Chromatography
Lab report
By–
Pratibha Chaudhari
9/28/2017
Introduction–
Affinity chromatography involves the property of bio–recognition for the separation of proteins based on the reversible interaction between the protein
and the specific ligand bound to a chromatography matrix. It enables us to purify bio–molecule on the basis of its biological function and its chemical
structure. The goal of the experiment was to gain hands–on experience in protein purification by using AKTA FPLC system. The IgG antibody
contained in the mouse antiserum was purified by protein G affinity chromatography using a HiTrap protein G HP column. The chromatogram was
then obtained and analyzed to ensure the separation of IgG antibody. Such purification of the protein before sample preparation is usually done to avoid
any undesired interactions.
Methods–
The IgG antibody was separated by using AKTA FPLC by setting the method as:– Column– HiTrap protein G HP 1 mL; Column pressure limit– 0.300
MPa; Flow rate– 1 mL/minute; Sample injection– 0.200 mL; Loading buffer, A– 0.05 M sodium phosphate, 0.15M NaCl, pH 7.0; Elution buffer, B–
0.1 M glycine–HCl, pH 2.7; Neutralization buffer, C– 1 M Tris–HCl, pH 9.0. Different column volume for individual step was then set as– 1 CV 0% B
wash and equilibration step; 1 CV 0% B loading; 4 CV 0% B washing; 10 CV 100% B eluting; 5 CV 0% B equilibration. After setting the method, the
system was washed with buffer B at the flow rate of

The Major Differences Between UPLC And HPLC
As previously mentioned the major difference UPLC and HPLC is the pressure and particle size. Pressures of up 1000 bar can be achieved with
UPLC, which is necessary to maintain shorter retention times. In UPLC smaller particles are used. These smaller particles provide a greater surface
area of the stationary phase within the column, making the separation of the substances much more efficient. However, the smaller the particles are, the
more pressure that is required. A summary of the main differences between HPLC and UPLC can be seen in the table below:
AttributeHPLCUPLC
Pressure6000 Psi100,000 Psi
Particle Size5Ојm1.7Ојm
Flow RateMillilitres per minuteMicrolitres per minute
Max ResolutionRelatively lowRelatively high
The lower particle size is the true reason for UPLC increased flow rate and resolution. This can be shown mathematically using Van Deemter's
equation: H = A + B/Вµ + CВµ. H being the plate height and Вµ being the particle size. The A constant remains constant independent of flow rate
(known as the 'Eddy diffusion term'). The B constant is the diffusion coefficient, and C is the "analyte mass transfer" coefficient. As Вµ decreases, the
A and C values ... Show more content on Helpwriting.net ...
Gumustas et al [3] reviewed that UPLC was more efficient than HPLC. In comparison to HPLC, UPLC can run higher resolution methods, using
shorter columns, smaller size of particles, with higher flow rates under high pressure. The injection volume of UPLC is almost ten times smaller than
that of HPLC, which results in good peak shapes and low carryover effects related to column diameter. It was also reviewed that the major
disadvantages of UPLC were the high prices of purchasing and maintaining the instrument as well as the shorter column life due to the increased back
pressure. However, the advantages of less solvent consumption, faster chromatography and better resolution properties overcome these

Glutathione S-Transferase Purification Lab Report
Lab Report: Glutathione S–transferase Purification Glutathione–S–Transferase Purification:
Two micro centrifuge tubes containing the mixture of 100uL Glutathione Sepharose 4B (collected from Prof Miguel) and 500uL of 1X PBS Buffer
(made by diluting 1mL of the 10X PBS buffer with 9mL of distilled water) was centrifuged for 1 minute. Removing its supernatant, it was
centrifuged again after adding 500uL of 1X PBS Buffer. To this, 300uL of "no IPTG" and 300uL of "+IPTG" Lysate samples (collected from Prof
Miguel) was added, which was incubated (while frequently inverting it) on ice for 20 minutes. After the incubation, it was centrifuged for 1 minute.
The supernatant from these samples of Sepharose 4B with "+ IPTG" and "–IPTG" was collected into ... Show more content on Helpwriting.net ...
30uL of the LB Buffer added to 30uL of each saved samples). Similar addition of LB Buffer (10uL) was made with the Protein Standards Ladder
(10uL). After the addition of the LB Buffer to the appropriate samples, it was placed on a 95oC hot plate for 8 minutes (note: it was instructed to
be for 5 minute). This was followed by the preparation of the gel setup (i.e. removing the tape at the bottom of the precast gel and adding the gels
at the back and front of the apparatus gel). To this setup, 50uL of 10X SDS running buffer + 450uL of deionized water (making it a 1X Buffer) to
the outer chamber was added. In the inner chamber, 150uL of 1X SDS (1X was made from the 10X SDS buffer, the same way by diluting it with a
1:9 ratio of deionized water) running buffer was added. With such setup and the 5 samples that was heated in 95oC, the lanes–starting with lane 1–were
pipetted in the following order: 10uL of Protein Standards sample (lane 1), 20uL of "+IPTG with Elution Buffer" (lane 2 and 3), 20uL of "–IPTG with
Elution Buffer" (lane 4 and 5), 20uL of "+IPTG Supernatant" (lane 6 and 7), and 20uL of "–IPTG Supernatant" (lane 8 and 9). After loading the gel in
such manner, the start of running the gel was done at 200V (making note of it being stable initially), and it was stopped till the bromophenol blue bands
reached ~1cm

E3 Agar Lab Report
Abstract: Through a series of experiments we are trying to demonstrate that we are able to express and purify a recombinant GFP in E. coli strain
BL21(DE3)<pRSETA–GFPUV using a Ni2+ agarose affinity chromatography method. The findings showed that one the highest amount of activity
came from elution 3 with 15144 RFU's. The estimated protein amount in Elution 3 was about 49ug using the Bradford assay, and specific activity of
309061 RFU/mg, the SDS page showed that E3s purity was about 33 %. The SDS Page showed faint bands at both, 28kDa and 32 kDa, and western
Blot analysis, showed that there was definite rGFP found in the E3, as well as all the Fractions. Introduction: Green Fluorescence Protein is a 238
amino acid protein, with a molecular... Show more content on Helpwriting.net ...
No water or SLB will be used, instead samples will now mix with Beta–Mercaptoethanol (2–ME) 6ul for G0 and G3, and 3ul for W3,W4, E2,and E3,
and 15ul of each (GCE, G3 and ladder only load 5ul) will be loaded into the gel. Run the electrophoresis at same voltage and time as last time. Once
the dye front is at the green gasket, turn off the machine, and pull the plates apart the same way take off the stacker, and notch the corner next to
the ladder. Set up the western blot machine, three wet filter paper, on the positively charged base, on top of that, a wet nitrocellulose paper then the
gel, make sure there are no bubbles, and three more wet filter papers on top of that, lastly the lid, which has the negatively charge base on it, lock
the lid, and allow to run over night. After the process is done, use forceps to take apart the layers of the blot, to reveal the nitrocellulose paper. Place
the paper in a Tupperware, adding 20 ml of Ponceau S stain, shake (rocking motion) for about 1–2 minutes discard the stain, and rinse the paper with
water, multiple times until red bands appear. Mark the ladder with pencil. Next place the paper in a blue Tupperware, with a clear lid, and add 30 ml
of 5% nonfat dry milk/ TBS solution, and place on the shaker for thirty minutes. When the time is up discard the Blocker (dry milk) and add 7ml of
mouse IgG anti–Xpress epitope Mab Solution, and place back on the shaker for 45 minutes. Once the 45 minutes are up, pour out the primary
antibody, and add 30ml of 0.05% Tween 20/TBS and place back on the shaker for five minutes, after, pour out the wash and repeat two more times.
Next, add 7ml of Sheep IgG anti–mouse IgG conjugated horse radish peroxidase polyclonal anti–serum solution and place on the shaker for 45
minutes. Once 45 minutes have passed, pour out the solution, and repeat the wash step from above, again three wash step. After the third

Salmon Lab Report
Raw salmon fillet was used as the sample for this experiment. Using a scalpel, 0.2 grams of salmon was incised and weighed in order to conduct
the experiment. The sample was transferred to a 1.5 milliliter glass tube for grinding. Using a pestle, the sample was ground into a fine paste for
pipetting. After the sample was ground, 300 microliters nucleic lysis solution, which was used to lyse the nuclei of the cells in order to obtain the
genetic material. The sample was ground again taking note of this imbalanced ratio of liquid to solid. Incubation at 65 degrees Celsius water bath was
administered to the freshly ground sample and nucleic lysis for 10 minutes. After the samples were warmed, the sample was centrifuged in a Bio–Rad
Model 16K Microcentrifuge... Show more content on Helpwriting.net ...
Subsequently, combination of 250 microliters PB buffer was added before vortexing for around 30 seconds for mixing. After mixing, the entire sample
was pipetted into the top compartment of a QIAquick spin container for continuation of purification. The sample was centrifuged at 14,000 rpm for 1
minute which allowed for the waste to flow into the bottom compartment which was discarded. This allowed the next buffer, PE buffer, to be added to
the top compartment. 750 microliters of PE buffer were added, and the mixture was again centrifuged at the same speed and for the same duration. The
liquid was again discarded from the lower compartment of the container. One more round of centrifugation then took place before the new buffer,
elution buffer (EB buffer), was added to the top compartment. 50 microliters elution buffer were added to the DNA sample and centrifuged at 700 rpm
for 3 minutes in a fresh test tube. The elution buffer eluted the DNA from the purification membrane and filtered to the bottom compartment of the
container during this centrifugation

Gapdh Lab Report
Abstract
Affinity purification is a powerful method of isolating a protein of interest from a complex mixture of proteins. the aim of this experiment was to
isolate the enzyme GAPDH from a complex mixture of proteins, in this case yeast lysate. in order to achieve this, a range of methods were used such as
protein, GAPDH activity and ADH assays, SDS–PAGE and imumunodetection of ADH and GAPDH on western blot. The results showed that the
isolation of GAPDH was achieved albeit, extremely with the % recoveries of the elution being abnormally high due to abnormally low initial material
values. The results suggested overall that isolation GAPDH from yeast lysate using affinity purification is in fact a somewhat effective method.
Methods
Affinity ... Show more content on Helpwriting.net ...
It was especially high in elution 1 and 2 who had values of 527% and 514% respectively, indicating a high GAPDH activity especially elution 1. This
is supported by the fact that elution 1 had the highest specific activity with a value of 0.0017 umoles/min/mg. it is however difficult to see in figure 2,
with the number of bands being seemingly lower than there should be.
The SDS–PAGE Electrophoresis (figure 1) contained different samples of the yeast lysate. From the gel the bands are clear and distinct with the
YNSMY–YNFT having the thickest and most distinct of columns making the cibracon blue an efficient tool for affinity chromatography. The protein
concentration from YNSM to YNW4 fluctuated with a general decreasing trend however the gel does not distinctively show this.
Limitations
The % protein recovery from the ADH assay was 556%, which could be due to a dilution error, most likely that the initial material had abnormally
low values. It was expected that elution 3 (YNE3) would have the lowest activity in GAPDH, from table 2 it is clear that it had the highest specific
activity but the lowest total activity and thus the lowest % protein recovery.
Overall, the results suggest that, this was somewhat of an effective method in isolation the enzyme GAPDH from yeast

Organic Chem 1 Post Lab Report Essay
Post Lab Report
Experiment 3 – Chromatography – Analyzing Analgesics by TLC and Isolation of ОІ–Carotene by Column Chromatography
Chemicals
1. Acetaminophen (C8H9NO2)
2. Aspirin (C9H8O4)
3. Caffeine (C8H10N4O2)
4. Ibuprofen (C13hH18O2)
Introduction
In this experiment, several analgesics were analyzed by Thin Layer Chromatography (TLC) and the composition of an unknown tablet was identified.
We define chromatography as the separation of two or more compounds or ions by their molecular interactions by either a moving or a stationary
phase.1 There are different types of chromatography: Thin Layer Chromatography (TLC), Gas Liquid Chromatography (GC), and Column
Chromatography (CC). All of which there two phases: ... Show more content on Helpwriting.net ...
Results
Thin Layer Chromatography Compound| Distance (cm)| Acetaminophen| 4.7| Caffeine| 1.5| Aspirin| 0| Ibuprofen| 0| Solvent front| 6.5|
Thin Layer Chromatography with unknown Compound| Distance (cm)| Unknown| 1.5|
Thin Layer Chromatography comparison Compound| Distance (cm)| Residue| 0| Isolated ОІ–carotene| 0| Standard ОІ–carotene| 0|
Calculations
Rf = Distance traveled by the compound / Distance traveled by solvent
Rf Acetaminophen = 4.7cm / 6.5cm = 0.7

Rf Caffeine = 1.5cm / 6.5cm = 0.2
Rf Aspirin = 0cm / 6.5cm = 0
Rf Ibuprofen = 0cm / 6.5cm = 0
Rf Unknown = 1.5cm / 6.5cm = 0.2
Discussion
The experimental results indicate that the identity of the unknown tablet was determined by measuring and comparing the distance traveled by the
known standards to the unknown standard. The Rf value of Caffeine was 0.2 and of the unknown was also 0.2 as shown by the calculations above,
this proves that the unknown substance was Caffeine, given that the unknown substance traveled 1.5cm and was equal to the distance traveled by
Caffeine, showing that they were the same. Acetaminophen had a Rf value of 0.7 with Aspirin having a Rf value of 0 and Ibuprofen also having a Rf
value of 0. This shows that the unknown tablet was Caffeine.
Yes, the

cell bio homework 3 Essay
Cellular Biology Lab – Homework #3
Due the week of Nov. 4th
You may use the lab manual, pre–lab lectures, and credible internet resources, however you may not use your cell bio lab classmates as a resource.
You will most likely see this material again on the Final and I highly encourage you to work individually and seek help from myself or your TA.
Plagiarism will result in an automatic zero.
1. In the cell bio lab, we use company manufactured gels, however you can make you own polyacrylamide gels. List all of the ingredients found in an
SDS–PAGEgel. Which ingredients are responsible for polymerizing the solution? How does the percentage of acrylamide effect the migration of
proteins (ex: 4% gel vs. 18% gel)?
The percent ... Show more content on Helpwriting.net ...
In this gel above, which lanes contain the positive and negative controls? Describe the information you can deduce by comparing and contrasting the
negative control and lane 2 protein bands. Lane 3 is the positive control and lane 4 is the negative control. By comparing lane 4 the negative control
and lane 2 the purified protein X it is noticeable that the second band in lane 2 is the elution buffer due to the fact that the negative control only
contains elution buffer and only forms one band which matches up with the second band in lane 2.
c. Describe the information you can deduce by comparing and contrasting the positive control lane and lane 2 protein bands. By comparing lane 3 the
positive control to lane 2 the purified protein X it is noticeable that the first band is the unknown protein X due to the fact that the positive control only
contains purified protein X and only forms one band which matches up with the first band in lane 2.
d. You don't know the molecular weight (MW) of protein X and you are not able to find that information in the scientific literature. The best way to
determine the MW of a protein using an SDS–PAGE gel is to use the protein ladder bands to create a Log(MW) vs. Rf graph and calculate the MW
from the line of best fit. What is the equation to calculate the Rf of a protein band? Make a table of the Log(MW) and the Rf values for all 5 protein
ladder bands. Describe any trends you see in the table

Immobilized Metal Affinity Chromatography
Proteins with his–tags are purified using IMAC, immobilized metal affinity chromatography. The protein product interacts with a metal that is
reversibly bound to an immobilized chelating group. The immobolized chelating group acts as a Lewis base (electron–pair donor) to which the Lewis
acid (electron–pair acceptor) metal ion is coordinated. The support to which the metal ion binds is called a ligand. When an electron donor group is
replaced by another, the action is referred to as ligand exchange. The donor atoms involved in this exchange are the electronegative nitrogen, sulfur, or
oxygen. These atoms scavenge for sources of electrons. The structure formed when the metal ions are added to form the chelate result in free
coordination... Show more content on Helpwriting.net ...
In this paper I will refer to the immobilized Lewis base as the ligand. The combination of the metal ion with the ligand will be referred to as the
affinity ligand. IMAC can be performed employing ion exchange resins as the ligand, but in the separation of proteins only chelating groups have
been used to fix the metal ion to the support. Schmuckler compared the binding energy of transition metal cations with ordinary cation exchangers
and with chelating groups. He found the chelating groups' binding energy to be 15 to 25 kcal/mol whereas the ordinary ion exchangers have a
binding energy of 2 to 3 kcal/mol. (Nes, 1999) This characteristic has led to chelating groups as the ligand of choice in IMAC. The chelating groups
used in IMAC are multidentate chelating compounds providing the strength of the complex formed by the protein, metal ion and chelating group. Free
coordination sites in the metal ion must be present in the structure formed after the metal ion is chelated by the chelating group to allow for the
adsorption or binding of solvent molecules or proteins. (Sulkowski, 1985) Differences in the number of free coordination sites plays a part in the
selectivity of chelating substances for a target protein.
Commonly used metal chelating substances include iminodiacetic acid (IDA) and nitriloacetic

Dimethyl Methyl Polysiloxanes
Column one was a 30m x 0.25mm–i.d coated with 0.5 Ојm 5% diphenyl and 95% dimethyl polysiloxane (RtxВ®–5). Column two was a 30m x
0.25mm–i.d coated with 0.5 Ојm midpolarity phase consisting of 50% phenyl and 50% dimethyl polysiloxane (RxiВ®–17sil). Column three was a
30m x 0.25mm–i.d, capillary coating with 0.5 Ојm film of 100% trifluoropropyl methyl polysiloxane (RtxВ®–200). The separation was performed
using the same temperature program on all these stationary phases. The temperature program used for separation was consisted of an initial
temperature hold at 80 В°C for 1.0 min, ramped up to 300 В°C at a rate of 30 В°C/min, held at 300 В°C for 0.5 min then ramped to 340В°C at a rate
of 5.0 В°C/min and held at 340 В°C for 5.0 min with a total run 21 min. (TP1). Column four was a 30m x 0.25mm–i.d, capillary coated with 0.5 Ојm
film of midpolarity phase consisting of 35% phenyl and... Show more content on Helpwriting.net ...
There is an extensive overlap and co–elution of the JWH–018 and 5–(1–naphthoyl)–1–pentylindole (compound 4) on Rtx–200. The separation is
completely resolved with full baseline resolution on Rtx–5 and Rtx–17Sil. However, the last four peaks showed slightly tailing but this tailing does not
affect the quality of the separation. On the other hand, the run time was increased on the Rxi–35Sil column to allow for the complete elution of sample
components. It takes more than thirty minute to get a complete separation with slow upward baseline shift. Otherwise, the separation was excellent
with slightly tailing in the last four compounds but this tailing does not affect the quality of the

Dna Isolation Research Paper
DNA Isolation, Quantification, and Visualization
DNA isolation is a process of DNA purification through physical and chemical means. DNA was protected within the nuclear membrane of B. rapa,
which was surrounded by a cell membrane and cell wall. Mechanical and chemical lysis of the cell was necessary to break open the cells and
solubilize the membranes to isolate the DNA. Mechanical lysis, which consisted of grinding the leaves with a pestle, broke down the cell wall.
Chemical lysis, through the use of lysis solution, broke down the lipid–base membranes. The lysate was placed inside the Zymo–spin IV spin filter to
remove chunks of debris from the solubilized cell components. A binding buffer was added to the collection tube that held DNA,

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

Ion Exchange Chromatography Lab Report
Abstract: Ion exchange chromatography is used to separate molecules base on their electrical charge and interpret the behavior of charged molecules
in different samples. This is done by using a 2–step salt gradient, during the separation, molecules were released from the column base on how strong
they are, the weaker substance was released first. While, in the gel filtration molecules are separated base on their size and shape using a matrix
porous which allows larger molecules to be release in the beads more easily than smaller molecules. Introduction: The aim of this experiment was to
observe how different procedures can be used to separate molecules base on their unique properties; such as, their electrical charge as well as their size
and shape. In the ion exchange, separation is done using an equilibrium of the molecules, any disturbance in the equilibrium will greatly affect the
strength and ph. of the molecules. Gel filtration, on the... Show more content on Helpwriting.net ...
Methods (Packing Column); mix the matrix, use 5 or 10ml pipet to pour matrix into the column, add buffer to fill the reservoir, put empty beaker under
column, remove cover from the column and allow the buffer to fill the column for about 10mins, and then cover the column. Packing is complete when
the matrix stops compressing at about 3ml. (Fraction Collection); label 8 test tubes (1–8) make sure to label them, remove all buffer from the above
bed with pipet, add the sample to the top of the bed using a pipet, place beaker under the column and remove cap from the spout, when the sample is
completely in the bed replace the cap, add buffer, continue to add buffer until the blue dye has reach the bottom then begin to collect 0.5ml fractions.
As the dye separates add fresh buffer, continue to collect 0.5ml in each of the 2–8 tubes. After all the tubes have been collected replace cap onto the

Ni 2 + Agarose Chromatography Lab Report
In this experiment the purpose was to see if E. coli could express a his–6 tagged recombinant Green Fluorescent Protein (rGFP) in a bacterial culture.
Purifying the sample through Ni 2+ Agarose Chromatography and then discovering the total protein yield through Bradford Assay determined
expression of his6 tagged recombinant rGFP in E. coli. The purity of rGFP in the samples was examined and the strength of the bands that appeared
at about 34.0 kDa (the MW of rGFP) in association to the ladder lane of the SDS–PAGE gel. Then a Western Blot was performed for the comparison of
rGFP bands to the ladder to reasonably determine the molecular weight of rGFP. It was determined that the purity of the band of E2 was at 80 %,
which made the calculated... Show more content on Helpwriting.net ...
The experiments that were completed previously offered a comprehensive understanding of how rGFP was induced, expressed, and purified. To outline,
Ni2+–agarose affinity chromatography was done to separate the protein of interest through a strong affinity to the His–6 tag in the rGFP to the column.
The Bradford assay is where the estimation of the amount protein of the samples was done. Then the SDS–PAGe gel showed an estimation of the
molecular weight and purity of samples. This was important in identifying the protein. Finally developed a Western Blot, confirming the presence of
rGFP through band

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