Glutathione S-Transferase Purification Lab Report

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 ...
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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
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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

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

Organic Chem 1 Post Lab Report Essay
Post Lab Report
Experiment 3 – Chromatography – Analyzing Analgesics by TLC and Isolation of β–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:
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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 β–
carotene | 0 | Standard β–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

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

Dimethyl Methyl Polysiloxanes
Column one was a 30m x 0.25mm–i.d coated with 0.5 μm 5% diphenyl and 95% dimethyl
polysiloxane (Rtx®–5). Column two was a 30m x 0.25mm–i.d coated with 0.5 μm midpolarity
phase consisting of 50% phenyl and 50% dimethyl polysiloxane (Rxi®–17sil). Column three was a
30m x 0.25mm–i.d, capillary coating with 0.5 μm film of 100% trifluoropropyl methyl polysiloxane
(Rtx®–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 °C for 1.0 min, ramped up to 300 °C at a rate of 30 °C/min, held at 300 °C
for 0.5 min then ramped to 340°C at a rate of 5.0 °C/min and held at 340 °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 μm 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,

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–Spin™ 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

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%

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 R² = 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 R² = 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 rGFP gene that
produces rGFP mRNA, which is then translated to produce the rGFP. By inducing the IPTG
activation of the DNA 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
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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

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 myoglobin〖

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

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

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

Synthesis Of Lycopene
Conclusion The isolation of β–carotene from lycopene via column chromatography was
accomplished, but results were inconsistent. Many students were unable to isolate the first elution
compound, β–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 β–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

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

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 μl'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 μl 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 μl 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 μl 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 μl 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 μl).
Once the elution had been completed, the sample obtained was further analyzed using a Nanodrop
2000 Spectrophotometer. The purity and concentration of the

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
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.,

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–PAGE gel. 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

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 GFPuv
gene (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–β–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 120µg and the concentration of the
protein was 24.0 µg/µl, which was within the allowed range of error. An enzyme 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
the ion–exchange chromatography indicated that the unknown protein sample contained two
isozymes of the enzyme N–acetyl–β–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

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
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

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:
Attribute HPLC UPLC
Pressure 6000 Psi 100,000 Psi
Particle Size 5μm 1.7μm
Flow Rate Millilitres per minute Microlitres per minute
Max Resolution Relatively low Relatively 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/µ + Cµ. H being the plate height
and µ 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 µ 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

DNAAnd 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
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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,

Protein Chromatography
Protein properties are directly affected by their environment, which depends on pH, temperature,
solvent effects, and pressure. With these affects has come more desire for better techniques in the
purification and separation processes. These processes hopefully will prevent loss of biological
activity and denaturation. With ion exchange chromatography comes many advantages over column
chromatography such as union between the target protein and functional groups at a very quick rate,
exponential growth without difficulty, short processing times, no heating, no extreme pH range, and
a multitude of other things (Santos et al. 2012). Ion exchange chromatography in short is ideal due
to the low cost, longevity through harsh cleanings, and high ... Show more content on
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The first included Cibacron Blue Sepharose CL–6B, proteins, and egg white lysozyme. They tested
the proteins and egg whites binding ability to the Cibacron Blue. To make matters easier, they only
searched for absorption of pure proteins from 0.05 M Tris–HCl buffer, pH 7.2. This was achieved by
eluting the absorbed proteins with 1M KCl In in 0.05 M Tris–HCl buffer. The second type included
immobilized monoclonal antibodies and the absorption of E. Coli B–galactosidase against B–
galactosidase. This was chosen due to the simplicity of measuring the activity of B–galactosidase
that could then tell us the concentration of the antigen. The only downside to this approach was B–
galactosidase has a much higher molecular weight that most antigens commonly used by this
method. This means the large molecule will have trouble penetrating the pores in the immuno–
absorbents. This was done by immobilizing the antibodies on a silica–based adsorbent, which was
then added to PBS buffer. The buffer contained antibodies. The flask they were in was then shaken
for four hours, followed by removal of all the unreacted protein that was done by using a funnel
with sodium chloride solution. To wash the gel they used propionic acid and then stored the
adsorbent in PBS buffer. Finally to elute the absorbed B–galactosidase, they used 6 M urea in 20
mM Tris–HCl, 10 mM ethylene diamine tetraacetic acid, and 10 mM sodium Chloride, 0.1 M 2–
mercaptoethanol, pH 7.2 (Chase

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

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

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

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 37˚C for 20

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 1 Stage 2 (Cycle 33) Stage 3 95⁰ C 95⁰ C 55⁰C
68⁰C 68⁰C 4⁰C 2:0 min 30Sec 45Sec 1:30min 7:0 min ∞ Table. ... Show more content on
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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

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
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

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

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 40°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
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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–PAGE Gel Electrophoresis and BCAAssay A crude
whey sample and a purified *–lactalbumin sample were prepared for SDS–PAGE gel
electrophoresis by mixing 20 µL of each sample with µL 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, * µL 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

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
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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

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
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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

Glutathione S-Transferase Purification Lab Report

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Glutathione S-Transferase Purification Lab Report