i need a tutor to complete 3 results conclusion sections.pdf

i need a tutor to complete 3 results & conclusion sections for my lab
results
biochemistry discussion question and need an explanation and answer to help me learn.
i have performed a thermofluor assay and another enzyme assay in my lab sessions and I
need a tutor to complete my results section and conclusion about the results for me.
i will send over my results from the lab sessions for you to complete it and preform some
excel to get results.my lab session notebook is attached.
Requirements: fully answered and explained
E LAB NOTEBOOK CYRUS
LAB 1
12/09/22 Plasmid Transformation
Aim: Carry out two transformations with plasmids containing the gene expressing the wild-
type protein and mutant protein into E. coli competent cells and plate them out onto two
petri disease containing Luria Broth Agar media plus the antibiotic. Also preparing the
liquid LB media that we need to grow two cultures overnight.
Method:
Preparation of LB agar plates:
First, I labelled two petri dishes with AU6 with one labelled W and one M. Then stand the
molten agar tube on a rack near a sterile environment then add 50uL of Ampicillin to the
molten agar plate. I then closed the lid and mix by inverting three to four times. Also do not
create bubbles, when mixed pour half into each of the petri dishes then carefully place on a
bench to allow the agar to set.
The concentration of the antibiotic in the molten agar is 50mg/ml.
Transformation of plasmids into E. coli competent cells optimised for expression:
I located two 1.5mL microfuge tubes of 50uL of competent cells and put on ice for 2 minutes
to defrost. Then labelled the lid to one with AU6 M and to another AU6M one for wildtype
and one for mutant. Then I added 5uL of plasmid wild type to the competent cell tubes
labelled W and M then mix the tubes by flicking 3 to 4 times and then place on ice for 20
minutes. After 20 minutes has passed, I used a heat shock both tubes by placing them in a
42 C heating block for 35 seconds. Then I transferred immediately onto ice for 5 minutes.
Afterwards added 250uL of SOC media to both tubes and place in the 37 C shaker/incubator

for 45 minutes. After 45 minutes you will plate out the SOC media+ cell mixture using a
sterile spreader. Do each mixture one at a time. Place the transformed petri dishes into the
37 C incubator overnight.
Preparation of LB media
I weighed out 1.25g of LB media and add to a 150 mL conical flask and then repeat to
another conical flask then add 50 mL of dH2O to both conical flasks. Then I added a foam
bung and a piece of aluminium foil to seal both flasks. Allow to sit for 5 minutes before
turning upside to sit in the incubator.
Results:
Figure 1. Colonies made petri dishes on the left for wild-type and on the right for the mutant
Conclusion:
It is possible to transform plasmid DNA into E. coli using the heat shock method. Said
methodology is a classic technique of molecular biology which allows the insertion of a
foreign plasmid -or ligation product- into bacteria. We can divide the transformation event
into two steps of stages. Firstly, the DNA is taken up across the cell envelope (cellular wall).
Lastly, the DNA stablishes as a new and stable cellular element.
During the present experimental session, we were able to successfully add Ampicillin into
two molten agar tubes. The mixtures were added into two Petri dishes with Luria Broth
Agar, a mixture that allows bacteria to grow and transform. Transformation is the process
by which a foreign plasmid is inserted into a bacteria cell, thus providing a new genetic trait.
In our case, the we pretended to transform plasmids of wild-type protein AND a mutant
protein into E. coli. Plasmids are very convenient vectors of new genetic material because
the make it easier for the cell to express that gene. The antibiotic is added because without
it, the plasmid transformation into E. coli is inefficient –with only 1 out of 10,000 cells on
average being able to transform. The results of this sessions show that both the wild-type
and the mutant proteins were able to prosper in the medium containing the E. coli and the
Ampicillin. The photo shows that both cultures look similar, meaning that the size of the
population in both cases is similar as well. However, it is interesting to note that the culture
of bacteria transformed with mutant protein plasmids are slightly more viable in the
medium than the bacteria transformed with the wild-type plasmids. Although
understanding why a certain plasmid is more successful in the transformation stage, what is
important to note in the obtained results is that E. coli is indeed sufficiently competent to
take up the plasmids with the proteins of interest.
LAB 2
13/09/22
Preparation of buffers with high and low imidazole concentrations for use in affinity
chromatography. Analyse a sample of WT protein using its size exclusion chromatography.
Method:
Preparation of 25mL Buffer A:
Take a 50mL graduated beaker and make-up the total volume to ~20mL by adding 1250uL
of 1 M Tris pH 7.5 then 1500uL of 5 M NaCl and finally 1 M imidazole 250uL after adding all
three give it a swirl to mix. Now add dH2O until 20mL is the total volume and then check pH
level until 7.5 pH is achieved. After this is achieved add more dH2O until 25mL is the total

volume. Transfer buffer to a clean falcon tube. Label Buffer A AU6. After this check pH using
a pH strip. Then store in the fridge. To adjust the pH, I used NaOH and HCl.
Preparation of 25mL Buffer B:
Take a 50mL graduated beaker and make up a total volume to ~20mL by adding 1250uL of
1 M Tris pH 7.5 then 1500uL of 5 M NaCl and finally 12500uL of 1 M imidazole after adding
all three mix by swirling. Now add dH2O until 20mL is the total volume and then check pH
level until 7.5 pH is achieved. To adjust pH use NaOH and HCl. After this pH is achieved add
dH2O until 25mL is the total volume.
Evening session:
Setting up the overnight culture
We are going to set up two starter cultures using LB which has already been sterilized. One
culture to grow the wild-type and the other to grow the mutant protein.
Method:
Setting up the cultures for overnight culture:
We have to work as cleanly as possible to avoid contamination. Take the two 5 mL LB
sterilized in the 50 mL Falcon tubes and label one of them AU6 W for wild-type and the
other AU6 MT for mutant. To each tube add 5 uL of ampicillin to protein. Using a sterile
inoculating loop, take three colonies (individually) from the wild-type petri dish and
inoculate the 5 mL LB Falcon tube labelled W. Then do the same from the mutant petri dish
and place three colonies (individually) into the 5 mL Falcon tube labelled M. Then close the
lids and then incubate overnight in the 37 C shaker/incubator.
Size exclusion and chromatography system:
I was shown a size exclusion procedure executed by our professor of UvrB which allows us
to collect and arrange to store UvrB from size exclusion using the standards blue dextran,
BSA, Carbine Amylase and Ribonuclease. Then UvrB was inserted into the loop. Then we left
the system overnight to obtain results of the standards and the UvrB by size exclusion
chromatography.
Results:
Figure 2. pH strip showing pH level for Buffer A
Figure 3. pH strip showing pH level for Buffer B.
Figure 3.B) Graph showing results from size exclusion chromatography system for
standards and UvrB.
Figure 3.C) shows logMW against Ve/Vo and shows an equation to calculate logMW of UvrB.
Figure 3.D) shows Chromatogram results from shown practical by professor.
Conclusion:
During this session we used affinity chromatography as a purification method of the wild-
type protein. To do so, we set up an FPLC system, which is a technique based on the liquid
chromatography methodology. It allows to separate proteins and other biomolecules from
mixtures (also known as matrices) of high complexity and diverse composition. It is a
require step because, during the plasmid DNA transformation, several by-products need to
be removed. FPLC allows the fast, efficient, and selective separation of the biomolecules of
interest. To perform FPLC analysis Buffer A AU6 and Buffer B AU6 were prepared following

the instructions given. Buffer A AU6 was prepared adding 1.25 mL of 1 M Tris (pH = 7.5),
1.50 mL of 5 M NaCl, and 0.250 mL of 1 M imidazole. Afterward, approximately 15 mL of
dH2O were added and pH was verified using a pH meter. The mixture was then poured into
a graduated cylinder and enough dH2O was added to reach 25 mL. This mixture was then
poured to the properly labelled Falcon tube. Buffer B was prepared using the exact same
method, but in this case 12.5 mL of 1 M imidazole were added instead of 0.250 mL.
The FPLC was then used to separate the standards (Blue dextran, BSA, Carbonic Anhydrase,
Ribonuclease) and the UvrB. The retention volume in millilitres show that the structures
with the highest molecular weight have the least interaction with the stationary phase of
the column, thus advancing with the eluent and, hence, needed less solvent to leave the
column. Such is the case of Blue dextran. Its MW is 2,000,000, which makes it the largest of
all the standards. Since it is so big, it cannot interact with the small pores that make up the
stationary phase, thus remaining within the liquid phase (eluent). However, Ribonuclease,
with a MW of 13,700, has the highest Ve (71 mL). This structure is small enough to interact
with the pores of the column matrix, thus being retained by it and taking the longest time to
leave the column. That is why it requires the largest amount of eluent in the series. Using
the standards, it was possible to calculate and then plot a logMW against Ve/Vo graph and
derivate the equation to calculate logMW of UvrB. Ve/Vo is a relative measurement which
allows us to quickly determine which substance will require the largest amount of eluent to
flow across the column. The largest Ve/Vo belongs to the substance that takes the longest to
leave the column. And it also belongs to the structure with the highest MW value. With this
equation, it is possible to determine the expected MW of an unknown protein. All we have to
do is perform the FPLC for the unknown protein and measure its Ve. Since we know Vo, we
can determine Ve/Vo and, using the equation derived from the graph, we can determine the
MW of the unknown protein. The chromatograph shows the peaks and the areas of each
peak. The ratio peak area / total area is approximately 0.92, which shows that practically all
the sample was recovered.
LAB 3
14/09/22
Morning session:
Firstly, we are going to prepare buffers to use in Ion chromatography on Friday. Later we
will inoculate the fresh media using our starter cultures.
Method:
Buffer preparation for ion chromatography:
Firstly, we will need to label four falcon tubes with AU6. Then label one buffer_0, buffer_50,
buffer_100, buffer_400, buffer_1000. Then I added 1250uL of 1 M Tris pH 7.5 to buffer_0.
Then I added 1250uL of 1 M Tris pH 7.5 to buffer_50 and 250uL of 5 M NaCl. I then added
1250uL of 1 M Tris pH 7.5 to buffer_100 and 500uL of 5 M NaCl. Then I added 1250uL of 1
M Tris pH 7.5 to buffer_400 with 2000uL of 5 M NaCl and finally to buffer_1000 I added
1250uL of 1 M Tris pH 7.5 and 5000uL of 5 M NaCl. Then I added dH2O until the volume
was 20mL and transferred each into a small beaker to test the pH levels of each were 7.5,
using 2 M HCl to 2 M NaOH if needed to adjust pH levels to reach 7.5pH. Once pH 7.5 was
achieved I added more dH2O until the volume was exactly 25mL.

Inoculation procedure/ Setting up the expression culture:
(Preformed the following steps while taking a lot of care to prevent contamination)
First, I located both of my 150 mL conical flasks containing LB and then I located both 5 mL
overnight cultures (labelled W and M). I then went and added 50 uL of ampicillin to both 50
mL of LB (labelled W and M). Then I added 500 uL of the overnight culture labelled W to the
50 mL LB labelled W and then I did the same but I added 500 uL of the overnight culture
labelled M to the 50 mL LB labelled M. Then to mix them both I swirled them gently. Then I
took a 1 mL sample from each flask (W and M) and put them into two 1.5 mL cuvettes
labelled W and M respectively. Then I had to locate the spectrophotometer and check if it
was set to the optical density of 600 nm. Using the 1 mL sterile LB provided I recorded the
blank and then measured the optical density at 600 nm of both 1.5 mL cuvette samples
labelled W and M. Then I placed our inoculated flasks in the 37 C shaker/incubator.
Wednesday afternoon
Inducing expression of wild-type and mutant protein:
First, I took a 1000uL of our protein mutant and wild-type from our conical flasks and had
to hand it to two cuvettes to measure the optical density600 nm (OD600 nm). I kept
measuring the OD600 nm until the OD600 nm value was from 0.6-0.8 because once it
reached that absorbance, we then induced the wild type and mutant with IPTG. I measured
the absorbance every twenty minutes until the values were between 0.6-0.8 for the wild-
type and the mutant. After my absorbance reached 0.673 for W and 0.715 for M I added
IPTG to the conical flasks W and M. Once the absorbance reached 0.6-0.8, I kept the last
cuvettes used to measure the absorbance and transferred them to a 1.5 mL microfuge tube
and labelled one BIAU6W and BIAU6M. After transferring them into the microfuges I stored
them on ice to centrifuge later.
Western blot buffer preparation:
I added 50 mL of 1X PBS with 450 mL of dH2O into a 500 mL Duran bottle and labelled it 1X
PBS AU6 and this was the stock solution. Then I poured 50 mL of the 450 mL duran stock
solution into a measuring cylinder to transfer the 50 mL into a 50 mL falcon tube and added
2.5 grams of milk to the falcon tube to produce the blocking solution which was labelled WB
Blocking AU6. Once I added the milk to the flask I then vortexed it until fully dissolved and
then stored in the fridge once completed. Then I proceeded to make the washing solution 1X
PBS + 0.05% TWEEN20. I did this by adding 0.45 mL of 50% TWEEN20 to the 450 mL 1X
PBS and mixing by inverting the bottle. Afterwards I labelled this WB wash solution AU6.
After all this was completed, I took an empty falcon tube and weighed out 0.2 g of milk and
put it into the falcon tube for the antibody solution. Then labelled this flacon tube “For
antibody solution AU6” and placed it on my tube rack.
Harvesting cells from induced samples:
First i located my induced samples that were kept on ice in 1.5mL microfuges labelled
BIAU6M and BIAU6W. Then I centrifuged both the samples using a microcentrifuge at room
temperature for 5 minutes at full speed. After centrifuge was completed using a 1 mL
pipette i discarded all the supernatant left in the microfuges and discarded them into virkon
solution and put the pellets on ice. Afterwards I resuspended the pellets by adding
BugBuster Master Mix to each sample. I calculated how much to add by doing OD600nm/8

the OD600nm value for both were the measured optical densities for W and M of the initial
samples. Once I suspended the pellets, I incubated both BIAU6M and BIAU6W by leaving
them on a tube roller for 10 min at room temperature. Then labelling two BLUE Eppendorf
tubes BIWC-W and BIWC-M I place 10 uL sample from each pellet into their respective
Eppendorf tubes for use in later SDS-PAGE analysis. Then I centrifuged the remainder of
each cell suspension BIAU6M and BIAU6W using a microcentrifuge at 4 C at top speed for
10 mins. After 10 mins was up, I took another 10 uL sample of each supernatant into two
BLUE Eppendorf tubes and labelled them BICE-W and BICE-M which will be used in SDS-
PAGE Analysis. Then stored them in a freezer.
Results:
Figure 4. pH strips showing neutral pHs for buffers for chromatography
Figure 5. Shows the absorbance optical density measured for the Wild-type and Mutant
samples.
Conclusion:
During this session the goal was to perform two different activities. Firstly, we needed to
prepare the buffers needed for ion chromatography, and then, we needed to inoculate fresh
media using our starter cultures. Ion chromatography is yet another separation and
purification technique. In this case, ions and polar molecules separate from each other
thanks to their different affinities to the ion exchanger.
Each buffer solution needed to have neutral pH (7.5) and they all contained 50 mM of Tris.
The difference between them was the NaCl concentration, which ranged from 0 to 1000
mM. Once this was achieved, both ampicillin and culture were added to the conical flasks
containing W and M protein, careful to no mix the cultures. Optical density was measured
before saving the samples into the incubator. Figure 5 shows how the optical density grew
over time until the desired 0.6-0.8 range was met. To understand the results, we need to
remember that optical density allows us to measure the speed of the light through a
material. A higher OD value implies that the material is denser. In the case of our proteins, it
means that the biomolecule is growing and gaining more complex structures. The optimal
value is around 0.6 because if the OD is lower, then there would be not enough protein to
continue the study, and if the value is larger, then the proteins would be too thick would
make it difficult to separate them from the matrix and analyse them further.
LAB 4
15/06/22
The aim of today's experiment is to harvest the overnight cultures and the bacteria should
have been expressing W and M proteins. I will then harvest the cells and lyse them to
release their contents which will then be centrifuged to separate the soluble material which
if done correctly will contain W and M proteins. Then we will perform an SDS-PAGE and
western blot which will show the protein bands. I also will perform protein recovery and
purification steps by performing affinity chromatography which we will then attempt to
remove the his-tagged wild-type and mutant proteins by performing gravity flow ion
exchange chromatography to purify the protein samples further. I will be doing an
Immobilised Metal Ion Affinity Chromatography after which I will perform an SDS-PAGE.
Method:

Collecting my samples:
First i located my overnight cultures containing W and M proteins and measured the OD600
nm of both cultures but before I measured the absorbance, I had to blank the spectrometer
to obtain my results.
Harvesting the cells from the expression cultures:
I transferred the ~50 mL cultures into two different Falcon tubes labelled WAU6 and MAU6
and centrifuged them at 3,200 x g for 20 mins in the centrifuge. Then I discarded the
supernatant into the 1% Virkon but kept the cell pellet which I then added 3 mL of
BugBuster master mix to W and M pellets, so the cell membrane breaks and to degrade DNA
and RNA and release the soluble material within the cells. I then resuspended the cell
pellets using a pipette pipetting up and down until all soluble contents were dissolved. I
then added DNasel to each W and M. Which then were incubated for 10 minutes using a
tube roller at room temperature. Then pipetted 5 uL of each suspension into a BLUE 1.5 mL
microfuge tube labelled AIWC-W and AIWC-M. Which were then stored on ice. I then split
the 3 mL of each sample left into 2x2 mL microfuge tube with 1.5 mL in each of W and M.
Labelling them AIW AU6 and AIM AU6 which then were centrifuged using a microcentrifuge
at 4 C at top speed for 10 minutes. Using a 1 mL pipette I transferred the soluble material
into 2 x 15 mL Falcon tubes which I labelled AICE-W AU6 for wild-type and AICE-M AU6 for
mutant. Then proceeding onto pipetting 5 uL aliquot of each of the falcon tubes and
transferred into BLUE 1.5 mL microfuge which I labelled AICE-W and AICE-M then stored
into an ice box. Finally discarding the white microfuge tubes containing just the pellets.
Preparing and performing SDS-PAGE:
I had now prepared eight BLUE eppendorf tubes labelled AIWC-W, AIWC-M, AICE-W, AICE-
M, BIWC-W, BIWC-M, BICE-W and BICE-M. I located and gathered all my BLUE tubes to add
14 uL of dH2O and 8 uL of 4X SDS sample buffer which contains SDS and reductant. I
centrifuged all the BLUE tubes for 3 seconds to bring and heated all the samples using a 95
C heating block for 3 minutes afterwards I then centrifuged again to get samples ready for
SDS page and western blot. Once the staples were ready, I now had to prepare the 1000 mL
of 1X running buffer by diluting 50 mL of 20X Running buffer in 950 mL of dH2O. After I
prepared the buffer, I assembled the SDS-PAGE precast gels onto the BOLT Mini Gel Tank. I
had to remove the white tape at the bottom of the cassette before placing the cassette in.
After I placed the cassette into the tank, I added the 1X Running Buffer to the tank making
sure the wells of the gel were covered with buffer. Then I took 13 uL of each sample and
loaded them into the cell in the order of BIWC, BICE-W, AIWC-W, AICE-W, BIWC-M, BICE-M,
AIWC-M, AICE-M but then I loaded in the ladder. After which I loaded another 8 wells with
the same order as before the ladder. Then once all 17 wells were loaded, I connected the gel
tank to the power supply and ran electrophoresis at 200V until the blue dye edge reached
the green base of the gel. I then set the voltage at 200 and time at 24 minutes as instructed
by professor and pressed RUN. After the 24 minutes passed i removed the gel cassette
carefully from the tank and placed it on tissues. I then took my opening lever to gently break
the cassette and then I took the level and cut the gel in half at the ladder well so each half
contained the 8 samples and ladder. One half was stained with Coomassie and placed on a
rocking platform overnight to stain the protein bands. After the overnight stain I will

destain by removing the Coomassie and adding just enough dH2O to cover the gel. The
other half I took to my professor to perform western blot on the gel by assembling the
blotting sandwich in the cassette. Once completed we put it into the TransBlot Turbo
Transfer System for 7 minutes at 2.5 A constant (up to 25 V). Once the 7 minutes was up, I
returned and collected the membrane and placed it into a clean gel box and added the Milk
blocking solution I prepared the day before and then stored it in the fridge on a rocking
table overnight.
Thursday evening
Affinity chromatography followed by SDS-PAGE analysis Experimental procedure:
First, I located my two clear falcon tubes labelled AICE-W AU6 and AICE-M AU6 from my ice
box. Then located two PolyPrep columns which contain 0.3 mL of pre-washed NiNTA resin
with a yellow stopper to prevent resin from drying out. I labelled one column W used for
wild-type protein purification and one M used for mutant protein purification. Then I
labelled the falcon tube containing the W column FT-W AU6 and the falcon tube containing
the M column FT-M AU6 (FT stands for flow through). Then I took 5 uL of cell extract into
two separate BLUE 1.5 mL microfuge tubes and labelled the tubes AICE-W and AICE-M and
stored them on ice. Using a 1 mL pipette I transferred all the cell extracts for W and M
samples into the appropriate PolyPrep columns containing the NiNTA resin previously
labelled W and M. Afterwards I incubated the cell extracts with the resin in the column for
15 minutes occasionally pipetting the mixture up and down with a 1 mL tip to make sure
that the protein interact with the resin once completed I removed the yellow stoppers and
collected the flow through until no more liquid was coming out of the PolyPrep tubes and
placed the yellow stoppers back on and transferred the PolyPrep column to new 15 mL
Falcon tubes labelled W-W and W-M ( W stands for Wash). Then I removed any
unspecifically bound protein from the resin by adding 4 mL of Buffer A to the top of each
PolyPrep column and left for 2 minutes to incubate then I removed the yellow stoppers and
let all the liquid drip into the falcon tubes labelled wash. Once the drip stopped, I removed
the PolyPrep columns and placed the stoppers on. Then I transferred the PolyPrep columns
to two new falcon tubes and labelled them E1-W AU6 and E1-M AU6(E1 stands for Elution
1). I then eluted my 6xHis-tagged proteins from the resin by adding 300 uL of buffer B to the
PolyPrep columns and mixed slowly by pipetting up and down once and left to incubate for
5 minutes at room temperature. Once 5 minutes passed, I removed the yellow stoppers and
collected Elution 1 until no more liquid was dripping and placed yellow tips back on. Then I
transferred the PolyPrep columns into 2 x15 mL Faclon tubes labelled E2-W AU6 and E2-M
AU6 and repeated the steps for elution 1 until no more liquid was dripping and then put the
yellow stoppers back on. Then I took 10 BLUE Eppendorf tubes and transferred 5 uL aliquot
from FT-W and labelled Eppendorf tube FT-W AU6 then 12 uL aliquot from W-W labelled
blue tube W-W AU6 then 12 uL aliquot from E1-W labelled blue tube E1-W AU6 and then 12
uL aliquot from E2-W and labelled blue tube E2-W AU6 and did the same for the mutant
protein purification. After I added 7 uL dH2O to the FT-W and FT-M samples only and then
located the BLUE tubes labelled AICE-M and AICE-W from earlier and added 7 uL dH2O to
them. Then I added 4 uL SDS-PAGE loading buffer to all the samples (CE, FT, W, E1 AND E2
for both wild-type and mutant). Then centrifuged all the BLUE tubes for 3 seconds and then

placed them all in heating block at 95 C for 3 minutes and then centrifuged all tubes again
for 3 seconds. Then I followed the same SDS-PAGE I used earlier and loaded the wells in the
order Ladder,AICE-W,FT-W,W-W,E1-W,E2-W,AICE-M,FT-M,W-M,E1-M and E2- M then once
electrophoresis was completed I removed the gel from the cassette and placed gel into a
new clean gel box and put the Coomassie stain into gel box and placed on rocking platform
to stain gel overnight to show protein bands.
Results:
Figure 1 shows SDS-PAGE of Harvested Cells from expression cultures.
Figure 2 shows SDS-PAGE of Affinity Chromatography.
Conclusion:
The protein expression was induced using IPTG, but only when the samples had the
appropriated optical density value at 600 nm. The Western blot buffer was then prepared
and the cells were harvest before the induction of the samples. The samples were then
prepared for SDS-PAGE with the eight BLUE Eppendorf tubes prepared AIWC-W, AIWC-M,
AICE-W, AICE-M, BIWC-W, BIWC-M, BICE-W, and BICE-M. The electrophoresis technique
allows proteins to be separated depending on their weight and their net charge at the pH
we are working. The chromatograms show that most of the samples have sizes ranging from
70 kDa upwards. This is consequent with the OD values obtained in the previous
experience. However, the protein size range varies too much, thus making it difficult to
determine whether a given size is more prominent than other. However, there is a clear
difference between the SDC-PAGE performed in the harvested sample and the SDS-PAGE
performed after the Affinity Chromatography. The first shows a chromatograph which is
more diffuse, almost showing a continuum. However, after the sample is cleaned with an
Affinity chromatography, the bands are more separated. This means that the purification
technique does indeed help cleaning up the samples and simplifies its study. Considering
this, we can focus on analysing Figure 2, instead of Figure 1. In figure 2 we can clearly
compare the wild versus the mutant protein. AICE-W and AICE-M are practically identical,
which means that both proteins successfully transformed and extracted. The same result
appears for E1-W and E1-W, and E2-M and E2-W. However, the rest of the samples have
differences. This means that the structural differences between the wild and mutant protein
are enough to affect their interaction with the plasmids of E. Coli and thus produce
significant difference in the cell-extract. For example, W-W has a wider MW range and a
higher concentration than W-M, which is practically non-existent (that is, almost not W-M
was extracted from the sample). Such is the case also for FT-W and FT-M.
LAB 5
16/09/22
Today’s aim we will be purifying the protein W and M further by gravity flow ion exchange
chromatography and will assess the fractions by SDS-PAGE and then concentrate the
fractions containing the purified protein. We will then complete our Western Blot and work
out which fractions from IEC contain W and M proteins and the concentrate these.
Method:

A) Selecting samples from IMAC steps that contain W and M protein:
From my SDS-PAGE analysis yesterday I selected with samples contained my proteins. I
selected E1 and E2 from yesterday. I then combined E1 and E2 of the wild-type and
transferred into a Falcon tube labelled IMAC-W and then did the same for the mutant and
labelled IMAC-M. I aliquoted 12 uL of each pool into BLUE 1.5 mL microfuge tube and
labelled the tubes IMAC-W and IMAC-M respectively then stored on ice.
B) Preparation of the protein samples prior to IE chromatography:
I then adjusted the concentration of my protein that was in a buffer so that the salt
concentration is only ~50 nM before mixing the ion exchange resin. I did this by calculating
how much buffer_0 to add by following the equation CiVi=CfVf so what I calculated was
Vf=CiVi/Cf which was 300x600/50=3600 uL. Then to get how much to dilute the buffer I
had to follow Vdilution=Vf-Vi which was 3600-600=3000 uL. I then added 3000 uL of
buffer_0 to my protein sample.
C) Preparing the polyprep column for gravity flow chromatography:
I then located two PolyPrep coloumns resting in two 15 mL Falcon tubes. Each column
should have contained 500 uL of Q Sepharose fast flow ion exchange resin. I then labelled
one tube W for wild-type purification and one tube M for mutant protein purification.
D) Binding the protein sample to the IE resin:
I then transferred the diluted protein sample into each polyprep tubes respectively. I then
left to incubate for 5 minutes occasionally pipetting mixture up and down with a 1 mL
pipette to make sure protein interacts with the resin.
Collecting the flow through:
Then I removed the yellow stopper and quickly transferred the columns into the 15 mL
falcon tubes. The flow through is then collected at the bottom of the tube. When no more
liquid came out, I put the yellow stoppers back on the columns and the falcon tubes
containing FT were stored on ice.
Washing resin with very low salt buffer:
Then I transferred the columns into new falcon tubes labelled 50. I then added 0.9 mL of
Buffer_50 to the columns and left to incubate for 5 minutes. Once 5 minutes passed i
removed the yellow stoppers and collected the liquid into the falcon tubes labelled 50 until
no more liquid came out and placed the yellow stoppers back on. Then stored the falcon
tube on ice.
H) Washing the IE resin with the medium salt buffer:
I then transferred the Polyprep columns into new falcon tubes labelled 400-1 and added 0.9
mL of buffer_400 to the top of the columns and leave to incubate for 5 minutes. Then I
removed the yellow stoppers and collected the liquid until no more was left in the columns
and stored the flacon tubes on ice. Then I put the columns onto new falcon tubes labelled
400-2 and did the same steps and stored these tubes on ice was completed.
I) Washing the IE resin with high salt buffer:
Then I transferred the polyprep columns into new falcon tubes labelled 1000. I added 0.9
mL of Buffer_1000 to the columns and left to incubate for 5 minutes. Afterwards I removed
the stoppers and collected the liquid until no more was coming out of the column and
placed the tubes on ice and the yellow stopper back on the columns. I now ended up with

6x15 mL Falcon tubes per protein FT-W FT-M 50-W 50-M 100-W 100-M 400-1-W 400-1-M
400-2-W 400-2-M and 1000-W 1000-M.
J) Analysis of samples by SDS-PAGE:
Into separate BLUE eppendorf tubes I transferred the following samples from wild-type
purification and mutant purification:
12 uL aliquot from "FT-W'", label the Eppendorf tube "FT-W AU6/
12 uL aliquot from "50-W', label the Eppendorf tube "50-W AU6*
12 uL aliquot from "100-W', label the Eppendorf tube "100-W AU6
12 uL aliquot from "400-1-W', label the Eppendorf tube "400-1-W AU6
12 uL aliquot from "400-2-W", label the Eppendorf tube "400-2-W AU6
12 uL aliquot from "1000-W", label the Eppendorf tube "1000-W AU6
12 uL aliquot from "FT-M", label the Eppendorf tube "FT-M AU6
12 uL aliquot from "50-M", label the Eppendorf tube "50-M AU6
12 uL aliquot from "400-1-M", label the Eppendorf tube "400-1-M AU6
12 uL aliquot from "400-2-M", label the Eppendorf tube "400-2-M AU6
I then located the two samples IMAC-W and IMAC-M and added 4 uL of SDS-PAGE loading
buffer to all fourteen samples. I then centrifuge the samples for 3 seconds and then heated
all the samples at 95 C for 3 minutes in a heating block. Then centrifuged for another 3
seconds. Then I had to load my samples onto the gel in an SDS-PAGE Gel tank in the order
Ladder, IMAC-W, FT-W-50-W,100-W,400-1-W,400-2-W,1000-W, IMAC-M,50-M,100-M,400-
1-M,400-2-M AND 1000-M. Once all was loaded and my gel has run i had to stain with
Coomassie based stain as before.
Friday Afternoon
METHOD OF WESTERN BLOT:
Washing the membrane:
First, I located my sample and discarded the blocking solution in the sink and replaced it
with 50 mL washing solution then incubated it on the rocking platform for 5 minutes then
repeated this step twice.
Preparation of antibody solution:
I then prepared the antibody solution by adding 1X WB washing solution up to the 20 mL
mark on the Falcon tube containing the milk power labelled FOR ANTIBODY SOLUTION
AU6. Then I pipetted 10 uL of antibody into the 20 mL antibody solution Anti-polyHistidine
Peroxidase conjugate antibody. Then I incubated the membrane by adding the antibody
solution and left to incubate for 1 hour on the rocking table.
i) washing the membrane:
I then discarded the antibody solution once the hour was up and replaced it with 50 mL of
washing solution and incubated for 5 minutes on the rocking table and repeated this step
twice, so it’s been done 3 times.
I then needed to develop my western blot by taking a DAB tablet and Urea Hydrogen
Peroxide tablet and adding them into a 50 mL Falcon tube being careful not to touch the
tablets with my hand. Then I added 5 mL of dH2O and vortexed the tube until the pills were

fully dissolved. Now the SIGMAFAST DAB Substrate Solution was ready to use. I removed
the washing the solution from the membrane and replaced it with the SIGMAFAST Substrate
Solution and incubated on the rocking table. I stayed by it as the solutions works within 1-5
minutes so it needed to be removed quickly and replaced with 50 mL of dH2O. Then I
replaced the solution with the dH2O and took a picture of the membrane and SDS-PAGE for
my results section.
Identification and concentration of samples containing the purified W and M proteins
Method
Identification of purest samples:
I looked at my SDS-PAGE after it was stained and had to pull the samples with my protein
which for me was 400-1-W,400-2-W,1000-W,400-1-M,400-2-M and 1000-M and kept this
sample for next step.
Concentration of protein and storage:
I then located a 2x 4 mL Amicon MWCO 30 KDa concentrator for my wild-type sample and
mutant sample (W and M). then I added 1 mL of buffer_400 to the top of concentrator to
wash the concentrator membrane and centrifuged them at a speed of 3200 x g at 4 degrees
for 5 minutes. I then removed the buffer from the bottom of concentrator and the buffer
remaining above the membrane with 1 mL pipette. I then added my proteins to the top of
the membrane and centrifuged following the same speed as before. After centrifuging the
concdentrators I checked to see if the samples were below 250 uL as they were. Then i
labelled two 1.5 mL microfuge tubes for the purified proteins of the wild-types PAW-AU6
and PSW-AU6(where PAW stands for purified protein Autumn term wild-type and PSW for
purified protein Spring term wild-type). Then I labelled two 1.5 mL microfuge tubes for the
purified proteins of the mutant and labelled them PAM-AU6 and PSM-AU6 which have the
same meaning as the other tubes but just M for mutant instead. Then I had to split the wild-
type purified protein into the tubes equally, so I calculated by pipetting that I had 175 uL of
wild-type so 87.5 uL in each tube labelled PAW and PSW. Then I calculated the same way I
had 114 uL of mutant so 57 uL into each tube labelled PAM and PSM. Then my purified
proteins were stored into a -80 degree freezer for later use in autumn and spring term.
Results:
Figure 1 shows SDS-PAGE of wild-type and mutant protein in buffers.
Figure 2 shows Western blot of buffers as labelled above.
Conclusion:
Similar to the observations made in the previous experience, adding the buffer to the
protein samples improve their behaviour through the electrophoresis process. Likewise,
Figure 1 shows a very clean electrophoresis result. This is because all the previous steps
taken to improve the purity of the sample allowed the proteins to be clearly separated
according their charges and their mass. Figure 2 shows the result after adding Western blot
buffer, which was prepared as specified in the discussions. This technique uses specific
antibodies to identify the separated proteins, based on their size. It allows us to notice that
the largest proteins are: AWC-W AWC-M, and AICE-M. Likewise, AICE-W is the protein with
the smallest size, but also with the smallest range of sizes. Comparing the W and M pairs, we
observe a similar behaviour than in the previous experience. The AIWC samples are similar

for W and M protein, however, the rest of the pairs look different. This is due to the specific
provided by the western blot method, which allows to identify specific proteins from a
complex mixture of proteins. Only those recognized by the reactant are marked in the
chromatograph. It is thus interesting to note that, for example, while AICE-M and AICE-W
had similar MW and net charge, they are structurally different. Different enough, indeed, so
that the western blot study shows strongly different patterns on the chromatograph. We
can see the proteins are present in AIWC-W, AICE-W, AIWC-M and AICE-M.
LAB 6
03/10/22
The aim of today’s online lecture was to learn Bioinformatics. I was taught how to download
and use Chimera which was designed to be an interactive visualization and analysis of
molecular structures and related data. Today’s session is to understand how to use chimera
and preform Bioinformatics on our wanted molecular structure and view and save
snapshots of macromolecular structures. I also will be learning how to use PDBe and RCSB
to find out information about publicly available protein or DNA/RNA structures. I also will
use PDBsum to access pre run bioinformatics analyses and summaries of information
relevant to my PDB structure. Also, we will be able to predict the potential effect of
mutations on the protein structure and function using several publicly accessible
bioinformatics servers.
Firstly, I started the class by downloading Chimera and learning what it can do and help us
with along the course of SLRP and how to use and view structures using the software. I
started by viewing a protein structure and seeing how we can select and unselect specific
parts of the molecular structure (such as atoms, residues, and chains) to view and save
snapshots of it. I was shown how to open and view the structure using chimera and where
to open a local file. Then I was taught using the dropdown menu in Chimera how to view
atoms, residues, and chains in the molecular structure. We were shown this buy the
professor leading the online lecture and then were given a few minutes to play around and
get the hang of Chimera alone by ourselves.
After being shown how to use Chimera we were shown PDB (protein data bank) a website
which shows us all structures of proteins RNA and DNA. We were shown using the search
bar we enter our ID for the protein we want to view and then shown how it takes us straight
to the page with details about our wanted molecular structure which has been deposited.
There is also a lot of information that has been derived once the protein was deposited in
the data bank. The page shows us the basic information and links to papers and their
authors.
It also shows us matrices about how good the protein structure compared to all known
structures deposited in the data bank in a lovely colourful diagram. We can also then go to
wwwPDB to view the validation diagram in more details and even find reports done on our
selected molecular structure and view bioinformatics in a lot more detail in the report. Then
I was shown to use the 3D view on PDB which lets us interactively look at the structure in
3D. I was then shown PDBe and how it’s like PDB on how it gives us information on our
structure searching it similarly as PDB by entering the code in the search bar. Then I was
shown PDBsum and how it’s very useful and contains a lot of information similarly to PDB

and PDBe. Using PDBsum we were shown we can find out information about the protein,
ligands, and clefts of our desired molecular structure.
LAB 7
17/10/22
In today’s lab session we will be finding the protein concentration using two methods. First
method will be The Bradford Assay and the second method will be Direct UV absorbance.
Bradford Assay:
First, I took 9 microfuge tubes and labelled them A, B, C, D, E, F, G, H, and I. Then I located
my buffer 400 from the Boot Camp week of SLRP and BSA stock solution. I then added 50uL
stock of BSA to microfuge A and 0 uL of Buffer and placed it back on the rack. Then I added
60 uL of BSA stock to microfuge B and then also added 20 uL of buffer 400 and placed it
back on the rack. Then I took microfuge C and added 50 uL of BSA stock and 50 uL of Buffer
400 and placed microfuge C back in the rack. Then I took microfuge D and added 25 uL of
microfuge B and 25 uL of Buffer 400 and placed it back in the rack. Then I took microfuge E
and added 50 uL of microfuge C and 50 uL of buffer 400 then placed it back in the rack.
Then I took microfuge F and added 40 uL of microfuge E and 40 uL of Buffer 400. Then I
took microfuge G and added 30 uL of microfuge F and 30 uL of buffer 400. Then I took
microfuge H and added 10 uL of microfuge G and 40 uL of buffer 400. Finally, I took
microfuge I and added no stock solution but added 50 uL of Buffer 400. Then I took my
protein wild-type and mutant that was prepared during boot camp week and took two
microfuge tubes labelled them W4 and M4 to prepare my protein solutions. In the W4 tube I
added 4 uL of my wild-type protein with 12 uL of buffer and then for the M4 tube I added 4
uL of my mutant protein with 12 uL of buffer and these are my protein solutions. Once all
these were completed, I located my 96 well plate and started to pipette my stock solutions
and protein into the 96 well for each solution I pipetted it 3 times into the well just as
shown below.
I pipetted each solution 3 times into the 96 well plate and only 5 uL of each solution into
each well and then once completed I pipetted 250 uL of Coomassie G-250 to each well and
once completed left to incubate at room temperature for 10 minutes. Once completed I
loaded my plate into the plate reader and measured the absorbance at 595nm and got given
the output results of my 96 well plate. I calculated the mean absorbance for each sample
and subtracted the blank from them.
Results:
Table 1. Concentration and absorbance. Our main goal was to determine the unknown
protein concentrations of W4 and M4.
Figure 1. Plot of absorbance (y-axis) and concentration (x-axis).
To calculate the unknown values, we must understand that in the best-fit equation:
y = absorbance
x = concentration
0.4406 = slope
0.3944 = the y-intercept

Since this is a linear regression, the independent variable should be concentration because
it predicts the value of absorbance. However, to find out the concentration of W4 and M4,
we will plug their respective absorbances as y and solve for x. Thus, let’s rearrange the
formula:
x = (y – 0.3944)/0.4406
W4 = (0.724867– 0.3944)/0.4406
W4 = 0.750 mg/mL
M4 = (0.769833– 0.3944)/0.4406
M4 = 0.852 mg/mL
Conclusion:
Bradford's assay, named after the biochemist Marion M. Bradford, is a protein
quantification method used to determine the concentration of proteins in a given sample. It
is based on the principle of dye-binding, where a colored dye binds to the proteins in a
sample, and the intensity of the colored dye is then used to measure the amount of proteins
present. Bradford's assay is a simple and cost-effective method to quantify proteins and has
been used in a wide range of applications, from medical research to biotechnology.
The purpose of Bradford's assay is to accurately measure the concentration of proteins in a
sample. This is particularly important for research related to cancer, where precise protein
levels can be used to determine the stage of the disease and monitor its progression.
Additionally, accurate protein quantification can help researchers develop new treatments
for a variety of diseases.
In this experiment, we performed a serial dilution of the BSA stock and obtained the
respective absorbances for each one of the diluted samples. We plotted the known BSA
concentrations versus their respective absorptions and calculated the best fit line equation.
This equation was employed to calculate the unknown protein concentrations of W4 and
M4, which were 0.750 mg/mL and 0.852 mg/mL, respectively.
Bradford's assay is also used to determine the amount of contaminants, such as DNA and
RNA, in a sample. This is important for medical and biotechnological research, as it helps to
identify and remove contaminants that could interfere with experiments. In conclusion,
Bradford's assay is a simple, cost-effective method used to accurately measure the
concentration of proteins in a given sample.
In conclusion, the Bradford assay is used in a variety of applications, from medical research
to biotechnology, to help identify disease states and develop new treatments, as well as to
detect and remove contaminants that could interfere with experiments.
Direct Absorption using Spectrometer:
First, I located my buffer solution, sample proteins w and m and dH20. Then I went to the
spectrometer and set it up. I selected the option to measure UV-visible absorption
measurements on the home screen. Then I selected the 0.5 nm pathlength and set the
wavelength to 220 nm to 400 nm. Then I started taking measurements first I pipetted 2 uL
of deionised water and cleaned the nano configured spectrometer. Then I closed the lid and
re-opened the lid and using a clean lint free cloth I cleaned the sample area top and bottom.
Then I pipetted another 2 uL of water onto the sample area and closed the lid then recorded
a background. Then I opened the lid using the clean cloth I removed any liquid and then

added 2 uL of water again and closed the lid but this time recorded a sample measurement,
but it was just noise about the baseline. Then I opened the lid and cleaned the sample area
for this time I added the buffer 400 to the sample area then took a sample measurement.
Once measured I opened the lid again cleaned the sample area and then loaded 2 uL of
Wild-type protein and recorded the sample measurement. Then after cleaning the sample
area any leftover liquid, I then placed 2 uL of the mutant protein into the sample area and
closed the lid to now measure the mutant sample measurement. I then took the recordings
for each Wild-type and mutant proteins at 280 nm absorbance 260 nm absorbance and 340
nm absorbance.
Results:
Table 2. Wild-type and mutant sample absorbances at different wavelengths.
Figure 2. Absorbance at different wavelengths.
Conclusion:
Direct absorption spectrometry is a technique used to measure the absorption of radiation
by atoms or molecules. It involves passing a beam of radiation through a sample and
measuring the amount of radiation that is absorbed. The radiation is typically in the
ultraviolet or infrared region of the spectrum, and the sample is placed between two
mirrors, which absorb any radiation that is not absorbed by the sample. This allows for very
precise measurements to be taken, as even small changes in the sample can be easily
detected.
In the present experiment, we did not measure a standard curve with known
protein concentrations via direct measurement. Thus, it is unreliable to measure the protein
concentration using the Bradford’s standard curve since this would be an inconsistent
approach. However, we were able to investigate to measure different absorbances under
different wavelengths. We can see that 260 nm is the best option out of the three
wavelengths that were tested. Thus, if an experiment is conducted to measure the
concentration of proteins in these samples, we should do it using a wavelength of 280 nm.
LAB 8
31/10/22
Today’s aim will be to re-do the Bradford assay as done on the 17th of October.
Bradford Assay:
First, I took 9 microfuge tubes and labelled them A, B, C, D, E, F, G, H, and I. Then I located
my buffer 400 from the Boot Camp week of SLRP and BSA stock solution. I then added 50uL
stock of BSA to microfuge A and 0 uL of Buffer and placed it back on the rack. Then I added
60 uL of BSA stock to microfuge B and then also added 20 uL of buffer 400 and placed it
back on the rack. Then I took microfuge C and added 50 uL of BSA stock and 50 uL of Buffer
400 and placed microfuge C back in the rack. Then I took microfuge D and added 25 uL of
microfuge B and 25 uL of Buffer 400 and placed it back in the rack. Then I took microfuge E
and added 50 uL of microfuge C and 50 uL of buffer 400 then placed it back in the rack.
Then I took microfuge F and added 40 uL of microfuge E and 40 uL of Buffer 400. Then I
took microfuge G and added 30 uL of microfuge F and 30 uL of buffer 400. Then I took
microfuge H and added 10 uL of microfuge G and 40 uL of buffer 400. Finally, I took
microfuge I and added no stock solution but added 50 uL of Buffer 400. Then I took my

protein wild-type and mutant that was prepared during boot camp week and took two
microfuge tubes labelled them W4 and M4 to prepare my protein solutions. In the W4 tube I
added 4 uL of my wild-type protein with 12 uL of buffer and then for the M4 tube I added 4
uL of my mutant protein with 12 uL of buffer and these are my protein solutions. Once all
these were completed, I located my 96 well plate and started to pipette my stock solutions
and protein into the 96 well for each solution I pipetted it 3 times into the well.
Results:
Table 1. Concentration and absorbance for the second Bradford’s assay. Our main goal was
to determine the unknown protein concentrations of W4 and M4.
Figure 1. Plot of absorbance (y-axis) and concentration (x-axis).
To calculate the unknown values, we must understand that in the best-fit equation:
y = absorbance
x = concentration
0.4343 = slope
0.3884 = the y-intercept
x = (y – 0.3884)/0.4343
W4 = (0.7158-0.3884)/0.4343
W4 = 0.754 mg/mL
M4 = (0.878133-0.3884)/0.4343
M4 = 1.128 mg/mL
Conclusion:
In this experiment, we replicated the laboratory procedure that was conducted on
October 17th, 2022. Our results were somewhat similar – though the protein concentration
of M4 was quite different (1.128 mg/mL vs. 0.852 mg/mL).
The present laboratory’s findings are not as reliable as the ones obtained previously. Our
present R2-value was 0.9323, meaning that 93.23% of the variation in absorbance was
correctly explained by the concentration. In the previous procedure, the R2-value was
0.986, which meant that 98.6% of the variation in absorbance was correctly explained by
the concentration – thus, our previous model had a stronger predictive power.
The discrepancies in results indicate limitations. The main limitation that must be
accounted for is my pipetting technique, which is still improving. Pipetting error could have
resulted in these different findings, thus indicating that I might need to practice. An
acceptable Bradford’s assay curve should have a R2-value > 0.95, which was not the case.
Thus, further practice on my end is needed.
LAB 9
14/11/22
Todays aim is to perform a melting temperature using ThermoFluor assay. The unfolding of
a protein can occurs when it’s been heated past a certain point. So tonight, I performed this
assay to hopefully unfold my protein.

Method:
Preparing assay solutions of the wild-type and mutant protein and dye for the assay:
First I had to calculate how much protein I needed to be able to prepare a 50uL of a 0.5 mg
mL-1 solution by using CiVi=CfVf. I rearranged it to get Vi so we will use Vi= (Cf x Vf) /
Protein concentration which was 0.9 x 4 so it was 3.6 mg mL-1.
I calculated that I needed approximately 7 uL by doing Vi= (0.5 x 50) / 3.6 = 6.9 uLor
approx. 7 uL. Then I took a two 1.5 mL tube microfuge tube and added 7 uL of my wild-type
into one tube and 7 uL of my mutant into the other and then keep on ice. Then I added 2 uL
of Sypro Orange dye solution to each microfuge tube containing the wild-type and mutant.
Then I added 41 uL of buffer_400 to the microfuges containing the wild-type and mutant to
make a total volume of 50 uL in each microfuge. Then I mixed them carefully avoiding to
make air bubbles and ppippeted up and down with a 20 uL pipette, making sure to change
tips to avoid contamination.
Preparing the controls:
i) Positive (Lysozyme) Control:
I took a 1.5 mL tube and added 1 uL of dye and then 12 uL of 2 mg mL-1 lysozyme ( as your
positive control), and 12 uL of the positive control buffer, mixed as described before.
ii) Negative (Buffer) Control:
I took a 1.5 mL tube and added 1 uL of dye and then 24 uL of buffer_400, mixed as described
before.
Transfer the samples in the plate:
See figure 1 below to see layout of samples.
From each 1.5 mL tubes with the WT and Mutant assay solutions aliquot in appropriate
wells 25 uL of the mixture twice each. Then I transferred 25 uL to appropriate wells. Then I
placed wells on ice. Then I labelled my bootcamp id AU6 on the side of my wells. Then staff
loaded samples Thermofluor machine.
Results:
Figure 1. Results of plate reader for ThermoFluor Assay.
Conclusions:
A Thermofluor assay (TFA) is a technique used to detect and measure the thermodynamic
stability of nucleic acid molecules. The TFA is based on the principle of fluorescence
resonance energy transfer (FRET) and is used to study the interactions between nucleic
acids and their associated proteins. The technique is based on fluorescent labels that are
attached to the nucleic acid molecules. Upon heating, a change in the fluorescence of the

labels is observed, which is directly proportional to the stability of the nucleic acids. The
melting curve obtained from a TFA experiment is a plot of the fluorescence intensity versus
temperature.
The melting curve obtained from a TFA assay is used to analyze the thermal stability of
nucleic acids and the interactions of associated proteins. The fluorescence intensity of the
labeled nucleic acids increases as the temperature is increased, until the nucleic acids reach
their melting point. At the melting point, there is a sudden decrease in the fluorescence
intensity, which indicates the disruption of the hydrogen bonds between the strands. As the
temperature is increased further, the fluorescence intensity increases again, indicating the
complete denaturation of the nucleic acids. The first transition, from low to high
fluorescence intensity, is the transition from double-stranded to single-stranded nucleic
acids, and is referred to as the “melting point” of the nucleic acids.
The melting curve obtained from a TFA assay typically consists of two distinct phases. The
first phase indicates the stability of the nucleic acids and the proteins, while the second
phase indicates the stability of the nucleic acids alone. The transition between the two
phases is known as the melting point, and indicates the point at which the hydrogen bonds
are disrupted. The melting point is the most informative point on the melting curve, as it is
indicative of the thermal stability of the nucleic acids. The melting point can be used to
calculate the thermal stability of the nucleic acids, inferring whether they are suitable for
their intended use.
In the present experiment, we identified the melting temperature by plotting temperature
versus -dF/dT (derivative of fluorescence emission as a function of temperature). The
melting temperature (Tm) is the lowest part of the obtained curve – thus, it is
approximately 70 oC. Both the positive and negative controls did not show the same curve
pattern, which is partially expected. The positive control should have shown a similar curve
as the remaining samples because it should have contained the sample. Thus, a pipetting
error must have occurred and the negative control was pipetted into the positive control
well. This calls for attention in a future experiment, as these results would not be
considered valid in a real-setting experiment.
In addition to the melting point, the melting curve obtained from a TFA assay can also be
used to determine the binding affinity of the associated proteins. The binding affinity is
measured by the slope of the melting curve, which indicates the strength of the interactions
between the proteins and the nucleic acids. A high slope indicates a high binding affinity,
while a low slope indicates a low binding affinity.
Overall, our results indicate that the Tm is around 70 oC. However, we may not safely make
this inference since the positive control did not work as expected.
21/11/22
LAB 10
Today’s aim will be to determine the ATPase activity of UvrB through a coupled enzyme
assay.
Measurement of ATPase activity:
First, I had to prepare a 25 uL sample of Wild-type and mutant of UvrB at 0.15 mg mL-1. I
calculated the Vi by using the equation Vi= (Cf x Vf)/Ci to determine how much buffer_400 I

would need to add to make two microfuges of 25 uL of W and M UvrB.
Then I did the same calculations to see how much buffer_400 to add to make two
microfuges of 12uL samples of W and M of UvrB at 0.6 mg mL-1.
Preparing the Mastermix:
I took a centrifuge tube and started to pipette 846 uL of water, 126 uL of Buffer_400, 12.6
uL of DTT, 25.2 uL of ATP, 25.2 uL of PEP, 36 uL of PK, 18 uL of LDH and 18 uL of ssDNA to
make a total volume of 1107 uL of mastermix in the centrifuge tube.
Preparing the plate/strip for the plate reader:
I then got given a plate of 2 columns and 16 wells for pipetting our W and M UvrB and
mastermix before adding mastermix and NaDH to each well. I pipetted the volumes as
shown below into each well one coloum for W and one for M.
Once I was done pipetting all the above into the plate for the plate reader, I then gave my
plate to my professor to enter the plate reader and waited for my results.
Results:
Figure 2. ATPase activity for Wild-type of UvrB
Figure 3. Results of ATPase activity from plate reader for mutant UvrB.
Conclusions:
A coupled-enzyme ATPase activity assay is a laboratory method that measures
ATPase activity in a biochemical sample. The reaction involves the transfer of a phosphate
group from ATP to an enzyme, such as an ATPase, which releases energy. This energy is
used to drive other reactions, such as the synthesis of NADH from NAD+ and pyruvate.
NADH is then used as a marker to detect ATPase activity, as its production is proportional to
the amount of ATPase activity in the sample. The reaction can be described as: ATP +
enzyme → ADP + enzyme-bound phosphate + energy → NAD+ + pyruvate → NADH + H+.
NADH is then detected using a spectrophotometer, which measures the amount of light
absorbed by the NADH molecules. Thus, the presence of NADH allows for the detection of
decrease in ATPase activity.
When substrate concentration is high, enzyme activity is expected to increase. This
is due to the fact that enzymes work most efficiently when they have a high concentration of
substrate molecules to work with. As the concentration of substrate molecules increases,
the number of enzyme-substrate complexes formed increases, leading to an increase in
enzyme activity. The activity is only at its maximum when all there are no enzymes, which
in this case is ATPase, available.
In the present experiment, we observed that, in the absence of protein, ATPase
activity remained uniform and low. In the absence of NADH, the ATPase activity remained
high as the assay was not sensible to detect decreases in ATPase activity. At higher protein
concentrations, ATPase activity was initially higher than that from samples with lower
protein concentrations, as expected. Greater concentrations of protein are equivalent to
higher amounts of substrate – thus, enzyme activity is supposed to increase.
As a conclusion, our obtained results were as expected.

16/01/23
Todays aim is to perform a melting temperature using ThermoFluor assay. The unfolding of
a protein can occurs when it’s been heated past a certain point. So tonight, I performed this
assay to hopefully unfold my protein.
Method:
Preparing assay solutions of the wild-type and mutant protein and dye for the assay:
First I had to calculate how much protein I needed to be able to prepare a 50uL of a 0.5 mg
mL-1 solution by using CiVi=CfVf. I rearranged it to get Vi so we will use Vi= (Cf x Vf) /
Protein concentration which was 0.9 x 4 so it was 3.6 mg mL-1.
I calculated that I needed approximately 7 uL by doing Vi= (0.5 x 50) / 3.6 = 6.9 uLor
approx. 7 uL. Then I took a two 1.5 mL tube microfuge tube and added 7 uL of my wild-type
into one tube and 7 uL of my mutant into the other and then keep on ice. Then I added 2 uL
of Sypro Orange dye solution to each microfuge tube containing the wild-type and mutant.
Then I added 41 uL of buffer_400 to the microfuges containing the wild-type and mutant to
make a total volume of 50 uL in each microfuge. Then I mixed them carefully avoiding to
make air bubbles and ppippeted up and down with a 20 uL pipette, making sure to change
tips to avoid contamination.
Preparing the controls:
i) Positive (Lysozyme) Control:
I took a 1.5 mL tube and added 1 uL of dye and then 12 uL of 2 mg mL-1 lysozyme ( as your
positive control), and 12 uL of the positive control buffer, mixed as described before.
ii) Negative (Buffer) Control:
I took a 1.5 mL tube and added 1 uL of dye and then 24 uL of buffer_400, mixed as described
before.
Transfer the samples in the plate:
See figure 1 below to see layout of samples.
From each 1.5 mL tubes with the WT and Mutant assay solutions aliquot in appropriate
wells 25 uL of the mixture twice each. Then I transferred 25 uL to appropriate wells. Then I
placed wells on ice. Then I labelled my bootcamp id AU6 on the side of my wells. Then staff
loaded samples Thermofluor machine.
Results:
Conclusion:

06/02/23
assay.
Results:
Conclusion:
06/03/23
assay.
Results:
Conclusion:

i need a tutor to complete 3 results conclusion sections.pdf

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