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please write a lab report. I attached the guidelines and a sample
that you can follow. The topic is cloning. Please follow the same
format, but write completely different lab report.I have an old
lab report with the introduction removed, but you can still use the
results and discussion section as an example for formatting and content.
Make sure to follow the sample but write everything different

Title of Your Report
Be a little creative
“Bio366L Lab Report”
gets boring
Name
Bio366L – Section # Group #
Date
TA: Name of TA
Introduction – 5 points
This document shows you exactly how I want the lab reports formatted. You should try to follow
this layout as best as you can. I have included mock figures, tables and formulas to show you what
I am looking for when grading your data. Sticking to specific formats and page lengths are an
important part of publishing scientific data. In fact, most journals will not accept your work if it is
too long or formatted incorrectly. For this reason, sticking to the format and page length is worth
2 point on your total lab report grade. Grammar and spelling are also important for effective
communication. Since modern word processing software checks for both, please use these to
make sure your report is not full of errors. I will subtract up to 5 points off your total report grade
for bad grammar and spelling.
The format should be double spaced text in 12-point font with 1-inch margin all around. I
recommend using a serif font (like Cambria or Times), but I know you may have a limited selection
of fonts so any 12-point font that is legible is acceptable. You should have a section title that is
bold and in 18-point font before each section, just like the one that says ‘Introduction’ here. You
should have a title page (which doesn’t count against your page limit), where you also list the
names of your collaborators (the other people in your lab group if any). Your title page with
collaborator list (including yourself) is worth 1 point.
The page requirement for the Lab Report is 5 to 7 pages. Remember, the title page and your list of
references DO NOT count against your page limit. You may also have additional appendices at the
end of your report which do not count toward your page limit as well.
Your introduction should probably be around 1 – 1.5 pages in length. Here, you are demonstrating
you having a firm grasp on the background information of your topic of choice. Without showing
that you have command over sufficient knowledge first, the rest of your report would not elicit
trust in your reader. You could summarize IN YOUR OWN WORDS, the results of your literature
research. This is one of the best places to get in your references to primary literature too.
Wikipedia might be a good place to start your initial search, but actually comb through the
references listed in the Wikipedia article. Some of the reference listed could be primary sources
that you can use. Do not list the Wikipedia article itself as a reference. Within your introduction,
you should also state the purpose of your paper, such as 1) elucidating the properties of
something, 2) detailing the process of a procedure, 3) demonstrating a concept, etc.
You should clearly describe how the experiments you are about to discuss in detail will
demonstrate the concepts you just wrote about. This does not need to be in-depth, but you should
have two or three sentences devoted to this.
The introduction is worth 5 points in total for your first report:
• 3 points for background info about the experiments (some from primary
literature). History of cloning, the various techniques
• 1 point for referencing your research adequately (your lab manual should be the
bare minimum reference here).
• 1 point for stating the purpose of this whole experiment.
1
Materials and Methods – 3 points
This section is only worth 3 points because you can state that you followed the lab manual, which
pages you used, and reference it as a source (just like you did before). If you made modifications to
any step in the procedure, list those as well. In addition, this section should be written with the
mindset that if another person were to repeat your experiment, he/she should get the same
results as you did.
Results – 5 points
In your materials and methods, you should have listed the various experiments and test methods
you have performed. Here is where you provide the findings of each experiment. Present the data
as they are without bias and undue interpretation. Be conservative in your data presentation, and
only show what you need to in order to get your point across. Overwhelming amounts of poorly
formatted data are not only confusing; they will increase your page length and ultimately cost you
points.
Please pay attention to how I want your data formatted. You must label AND caption every figure,
table and/or formula. This is worth 1 point. If you need to, you may combine the Figure title and
caption together, but you must have a narrative accompany each figure and also reference your
figure in your text; instead of having a figure just floating in your report without a purpose.
Figure 1. Title this figure something descriptive that relates to your data. The title
should be bold, and 14 point font.
3.5
Axis Title
3
y = -0.9121x + 3.7839
R² = 0.4722
2.5
2
1.5
Y-Value 1
1
Linear (Y-Value 1)
0.5
0
0
1
2
Axis Title
3
Caption. This is where you
should write a descriptive
caption for your figure. You do
not need to discuss the
conclusions of this data, but do
list what the data is, what the
legend means, and what any
arrows or other symbols are
referring to. Also, for graphs
with trendlines, include the
formula and the R-squared
value.
2
Conclusions – 10 points
Here is where you get the majority of your points for the lab report. First, introduce each
experiment (part A, B, C…etc.) by discussing the objective of performing it (what are you trying to
show), and talk briefly about how you obtained the data (DO NOT reiterate the methods, just
describe the basic concept). This is worth 2 points. You do not need to include every piece of data
you obtained. What’s most important here is to show that you performed an experiment and
obtained data that either do or do not support the objective of said experiment.
If you used data that your lab group did not generate, you must reference your data source, and
explain why you did not use your own data. This is a good opportunity to demonstrate your
understanding of the experiment by describing what went wrong with your procedure. Your
discussion of the data is worth 3 points.
You stated your data as they are without bias in your results section, now is the time for you to
present your interpretation/analysis. What do all the data suggest? Synthesize a conclusion using
all the available data (including data from your background research). It is Ok if you have
conflicting data. State where you think the source of conflict might be and offer solutions. If your
data are unclear, then propose other experiments you might do to obtain clarity in your next
approach. Are there anything you can do differently next time to save your data from being
useless? All of this, plus relating the discussion back to your experimental objective is worth 4
points.
You should specifically state the questions asked in the lab manual at the end of each experiment,
and answer those questions by referencing your data and conclusions for that experiment. This is
worth 1 point. I expect to have a concise discussion of objectives, data, conclusions, and answers
to the lab manual questions (if any) for each experiment BEFORE you move on to discussing the
next one. This will make it much easier for me to grade each part of the exercise independently, so
if you missed a day or something went wrong with the next part, I can take that into account
separately from the ‘good’ data.
Heading 1
Data title 1
Data title 2
Heading 2
Heading 3
Heading 4
56.7
67.8
78.9
12.3
34.5
67.8
Table 1. Tables should be numbered
separate from figures. The title
should be bold, and 14 point font.
Table caption. This is where you should
write a descriptive caption for your table.
3
References – 2 points
This should be some easy points. You get 1 point for having AT LEAST 3 references, and 1 point
for having AT LEAST one primary literature reference. Primary literature means original, not
previously published research (reviews of lots of other people’s research don’t count). You can
find these by going to PubMed, Google Scholar, or using the library research tools. You may use
any reference style, but keep in mind the page limits. I recommend the APA citation style or the
number-in-line style, where you cite using a superscript number. 1 You will have to list your
references in numerical order though. Also, DO NOT cut and paste a link to a webpage as a
reference. MS Word has a reference manager that will allow you to input webpages as references.
If you are not using Word, look up how to appropriately reference webpages on the internet.
Points breakdown
Introduction
Materials & Methods
Results
Conclusions
References
Total
5
3
5
10
2
25
Other deductions
Format deviation (2)
Missing title page (1)
Missing label/caption on figures and tables (1 pt per occurrence)
Incorrect grammar (5)
4
Cloning: It Shockingly
Isn’t the Same Person
Zetouna 1
Introduction
Throughout a series of conducted experiments, fundamental cloning methods were used to
clone and amplify DNA with a target sequence accompanied by confirming the inserted gene, Tn10.
This gene codes for resistance to an antibiotic called gentamicin through a restriction enzyme digest.
A restriction digest of the plasmid with the HindIII restriction enzymes was performed. Such
procedures are essential in the molecular biology lab, as it lets researchers analyze and study target
genes or other DNA fragments of interest and even synthesize proteins. Certainly, such
bioinformatic technology and analysis in the lab could not have existed if it wasn’t for the
forefathers and their irreplaceable knowledge and commitment to the development of these lab
techniques. DNA cloning is a molecular biology approach that makes numerous copies of a piece of
DNA, like a gene. It spans back to the late 1950’s, when John Gudron from Oxford University
performed experiments with frogs which allowed him to produce cloned frogs from cells he
collected from a tadpole. This was the first somatic cell nuclear transfer experiment to be conducted
on frogs and it showed that “an adult nucleus could be reprogrammed to an earlier developmental
stage”- an extraordinary work that won Sir John Gurdon the Nobel Prize in 2012.1 This teaching was
passed on until 1996, when the most famous experiment was conducted- “Dolly the sheep”. Dolly
was the “first mammal to be cloned using a cell derived from an adult animal” and not an embryonic
cell1. A fascinating feature is that Dolly was “identical” much like monozygotic twins are “identical”
to the animal from which the cells were taken to create a cloned embryo. This describes a technique
known as reproductive cloning defined as a new animal “created” using genetic material from any
cell1. Another discovery they examined was that the telomeres of Dolly the sheep were shorter than
1
Christine Mummery, Anja van de Stolpe, Bernard A.J. Roelen, Hans Clevers, Chapter 6 – Cloning: History and Current
Applications, Stem Cells (Second Edition), Academic Press, 2014, Pages 131-161, https://doi.org/10.1016/B978-0-12411551-4.00006-4.
Zetouna 2
normal: “at one year of age the lengths of her telomeres were comparable to those of a six-year-old
sheep, suggesting that Dolly might age unusually quickly.”1 The world of cloning is endless and
there have been various techniques and methods that have evolved. In this lab, PCR amplification
was used in the cloning process. However, the existing various techniques in academic labs include
“the Gibson isothermal assembly method, the ligase cycling reaction (LCR), sequence and ligase
independent cloning (SLIC)…, and circular polymerase extension cloning (CPEC)”. 2
The use of restriction enzymes in the cloning experiment is significant to manipulating DNA
and aiding with insertion of genes into plasmid vectors. One possible way to make the final vector is
to glue the two ends of DNA together by using ligase which will seal it. However, ligase is
ineffective in positioning the two DNA ends together3. This is where sticky ends have the upper
hand since the overhangs are matching, they anneal and “stay” together for a sufficient time to have
ligase seal the nicks in DNA. Moreover, PCR was also used to intensify the gene of interest so it can
then be inserted into the vector of choice as well as ligated. 3
Lastly, clone verification must be performed to look at the DNA that was inserted into the
vector and distinguish if it’s the gene of interest. With the use of radioactive sequencing or
fluorescence labeling (used in this lab), it is simpler to see the gene of interest.3
The goal of this experiment was to clone a gene of interest or “digest” the final plasmid back
into the insert and plasmid to verify that cloning was done correctly. Additionally, the conducted
experiments show the various techniques by isolating the gene of interest, inserting it into a vector
2
Dalonso, Nicks & Savoldi, M. & França, P.H.C. & Reis, Thaila & Goldman, G.H. & Gern, Regina. (2017). Sequenceindependent cloning methods for long DNA fragments applied to synthetic biology. Analytical Biochemistry. 530.
10.1016/j.ab.2017.04.018.
3
Lee, Chi. 2018. Bio366L manual, San Diego State University 47:37-62
Zetouna 3
which replicates it, and cloning the gene of interest. Finally, the purified plasmid ran on a gel to
observe for accuracy, and if we saw it was right, the plasmid would then be sequenced.3
Materials and Methods
Our lab group followed the Biology 366L Manual of Fall 2020 on pages 37- 62 closely with
the methods “virtually” during these experiments. Most of it was performed by TA’s due to the
COVID-19 pandemic.
Results
Figure 1. Transillumination of successful PCR product after gel electrophoresis of
controlled gel
This is the controlled gel results of the pieces
placed into the bacteria. Group 1 shows the bands
with the dashed rectangles around them as the
bands to be cut out of the gel to continue the
experiment with (ligate the insert and the vector
together). The bands seen are ~1.5 kbp and ~3.0
kbp, respectively.
Figure 2- Gentamicin plates used to grow insert & vector in E.coli bacteria
These show the agar plates that contained heterogeneous cells
were purified. The ones with the highest number of dots grew the
most because of the Gentamicin resistant vector that they have
and the success it has at opening up the vector. Group 7, the
control plate had no growth since it was not digested, thus the
Zetouna 4
vector remained closed. It did not allow the insert to get into the
vector which led to no growth. Growth on the other plates means
the E. Coli successfully replicated the insert and plasmid.
Figure 3 – Successful transformation of Tn10 gene into transformant plasmid, seen by
transillumination after gel electrophoresis
This is the sample gel electrophoresis result in which we analyzed
the DNA from the bacteria with a gel. Since only Hind III was used
in the lab, the bands seen are 1490 bp and 2900 bp, respectively.
Figure 4. Tn10 Insert Sequence
The sequence below is the known sequence for the Tn10 insert. The sequencing results were aligned from the
two sequencing files from canvas; the forward primer (M13F-21) and the reverse primer (M13R) which were
aligned using www.expasy.org. Then, both were aligned separately to the known sequence for the Tn10 insert
(from a word doc) and the known gentamicin sequence (found on NCBI). The resulting sequence shows
annotation which indicates red letters as being verified using M13F-21 primer from the bottom up (reverse
Zetouna 5
complementary). The yellow highlight shows the sequence was verified using the M13R primer from the top
down. Finally, the bold & underlined sequence shows the gentamicin resistance gene from NCBI.
Discussion
The results are summed up next to the figures above, nevertheless, a further explanation of
them is to be followed in the conclusion. Figure 1 reveals the gel electrophoresis was performed to
observe and distinguish the different bands of DNA. Since the pieces were placed into the bacteria,
the goal is to pick one that has the most intense brightness and isolate the DNA following the
exercise 3 part D procedure. If this was done in the lab, students would have run four different
groups on the same gel. Thus, each of these groups on the gel is considered to be the same.
Zetouna 6
To simplify, figure 2 shows base pairs (bp) in their true size, and so, 3,000 bp correlates to
3kb; 1,500bp corresponds to 1.5 kb, et cetera. In the figure, the experimental sample in Hind III, as
compared to the ladder in the first lane, closely matches at 3 kb and 1.5 kb- naturally, visual
estimation through an image is not fully accurate. The expected values for the band sizes for Tn 10
vector was 1.4 kb whereas the expected value for pUC19 was 2.7 kb. The actual Tn10 resulted in a
vector of ~1.49 kp and the pUC19 being ~2.9 kb. Referring to figure 2, the sample gel
electrophoresis was done to confirm if the size of the insert/vector product was successfully cloned
in the bacteria that was grown. After growing the bacteria, the pieces taken out of the bacteria (figure
2), were seen to be the same pieces as the ones placed into the bacteria (figure 1). Since the
comparison matched, this means the bacteria did contain the insert/vector product and the
experiment was successfully cloned in the bacteria grown. Figure 3 shows the analyzed DNA from
the bacteria with the gel electrophoresis. The results of the bands seen are 1490 bp and 2900 bp,
proven to be closer to the expected values.
Finally, figure 4 shows the sequence of the DNA from the bacteria. The two sequences
known as the forward and reverse primers were aligned to the original Tn10 sequence. Aligning is
when the known sequence is taken and compared to an unknown sequence to see their similarities. If
they are exactly the same then the unknown sequence is discovered. For instance, the Tn10 and the
gentamicin sequences were known because they are published online. The goal was to compare
(align) sequencing results from the bacteria grown to those known sequences. If they align (if they’re
the same), then that means the Tn10 sequence and the gentamicin sequence was inside the bacteria
grown and the lab was successful, proven to be the outcome in this case.
Conclusion
Zetouna 7
Overall, in exercise 3 there were 7 parts (A-G). Part A included the preparation of the PCR
products done by the TA’s. Part B followed by the preparation of the vector DNA by using the
Invitrogen Purelink Quick Plasmid Miniprep Kit. Moreover, part C followed the running of the gel
electrophoresis of the restriction digests to fully separate them and see their size in base pairs. The
size was measured correctly, with the vector being ~2900 bp and the Tn10 gene being ~1500 bp.
These results can be found in Figure 2 above. Part D included following Appendix 5 to isolate the
vector and insert. Part E involved the ligation of the fragments from part D. Part F required
purifying and desalting the ligation from above so it could be electroporated. Lastly, part G included
the use of electroporation to transform the control and the ligation, which then were plated on LB +
gentamicin agar. The rest of parts A-G were completed with no changes occurring to them.
For Exercise 4, 6 parts were performed (A-F). Part A consisted of performing a restriction
digest of the transformant plasmids and preparing a gel with TAE buffer. Evidently, the colonies
survived in the group 7 plate since the control and group 7 plates were accidently switched. The gel
electrophoresis of the restriction digests was running in Part B. The ladder was set inside the gel and
electrophoresis was running with the restriction digest. Part C involved staining the gel, part D was
analyzing the gel with the naked eye. Part E part E was the submission of samples for sequencing.
All in all, the first gel was done to purify the insert and vector to ligate them together and the
second gel was used to verify that the bacteria grown actually took in the plasmid with the
insert/vector. According to the presented data, the lab was conducted steadily with little errors. This
reveals that our DNA clone was verified because the insert consisting of gentamicin resistance was
Zetouna 8
inserted in the plasmid. The insert was shown in the gel through evidence; the first column on the
right of the ladder was the uncut vector with no HindIII enzyme added to it, but the second column
shows the digested vector with HindIII enzyme. After the bacteria grew, the pieces taken out of the
bacteria (figure 2) were analyzed. The results prove the DNA clone was verified since the presence
of the insert (selected band) was a little over 1400bp. Thus, the experiment was successful because
the same bands were seen in both of the gel electrophoresis figures. According to figure 4, the
sequencing results from the bacteria aligned with the Tn10 sequence and the gentamicin sequence
show a clear indication of how Tn10 and gentamicin sequence were successfully cloned into the
bacteria. The sequence was included as a figure so that experimenters who want to replicate the
experiment can see the insert and primers that were designed.
Pertaining to the manual’s further questions, the gels reveal the expected values since they
are close to each other numerically. The PCR (Tn10) insert was 1490 bp long, close to the expected
1400 bp insert band. Similarly, the pUC19 vector is 2900 bp, also near the expected 2700 bp. Thus,
the bands approximate to those lengths as compared to the ladder in the gel meaning the experiment
was successfully conducted and the vector was preserved.
Zetouna 9
References
Christine Mummery, Anja van de Stolpe, Bernard A.J. Roelen, Hans Clevers, Chapter 6 – Cloning:
History and Current Applications, Stem Cells (Second Edition), Academic Press, 2014,
Pages 131-161, https://doi.org/10.1016/B978-0-12-411551-4.00006-4.
Dalonso, Nicks & Savoldi, M. & França, P.H.C. & Reis, Thaila & Goldman, G.H. & Gern, Regina.
(2017). Sequence-independent cloning methods for long DNA fragments applied to
Zetouna 10
synthetic biology. Analytical Biochemistry. 530. 10.1016/j.ab.2017.04.018.
Lee, Chi. 2018. Bio366L manual, San Diego State University 47:37-62
Lactate Dehydrogenase Purification Report
Kyle Malter
153L, 1H
Ta: Joe Cao
Use these formulas for the following results
Specific Activity is
% recovery is
Fold purification is:
1
2
Results:
Ammonium Sulfate Fractionation was used to separate proteins based on their
3
hydrophobicity. Hydrophobicity of LDH was determined by the proteins primary sequence and a
4
hydrophobicity plot generated using the expasy program (Figure 1). After determining the
5
solubility of our protein, the two-step salting out procedure was determined to be . First a 40%
6
Ammonium Sulfate step would be used to remove highly hydrophobic membranes and
7
organelles, leaving the more soluble protein in the supernatant. This step would then be followed
8
by a 60% precipitation of LDH and subsequent resuspension. Our data shows an 86 ± 1% total
9
LDH recovery from the supernatant of the 40% Ammonium Sulfate step, when compared to the
10
crude lysate. This was followed by a 63 ± 2% total LDH recovery in the following 60% salting
11
out step.
12
In order to determine the relative amount of LDH recovered, we preformed enzymatic
13
assays of the two recovered fractions following each step. Our 40% salting out step resulted in
14
4920 ± 20 relative activity units for the recovered fraction. The 60% salting out step resulted in
15
3590 ± 60 relative activity units. Using these data and our previous total protein data, we could
16
calculate the relative specific activities (RSA) for both fractions. The RSA was used to confirm
17
presence of LDH via enzymatic activity and compare relative concentrations of LDH vs
18
contaminating peptides. Our 40% fraction resulted in a RSA of 29 ± 1 (units/mg) and our 60%
19
fraction contained a RSA of 57 ± 2 (units/mg). The fold change in purity for our 40% step was
20
0.99 ± 0.07 vs crude and for the 60% step was 2.0 ± 0.1 vs crude.
21
Taken together these data show that the 40% purification step did not increase the purity
22
of LDH vs the crude lysate. However, the 60% salting out step resulted in a two-fold purity of
23
the enzyme.
24
After recovering the pellet from the 60% Ammonium sulfate purification, the
25
resuspended pellet was purified by affinity chromatography using an AMP-agarose resin matrix.
26
Fractions were collected and assayed for both total protein concentration and enzymatic activity
27
of LDH. The fractions were separated by load, wash and elution, based on the buffers run
28
through the column. Our results showed a large increase of proteins during the load phase of
29
purification (figure 2). However, the enzymatic activity of LDH was relatively low indicating
30
most the unbound proteins were contaminating proteins. Our wash steps showed no increase in
31
either protein nor enzymatic activity. The elution fractions 10 and 11 contained the most
32
enzymatic activity of all the fractions with 1321.543 Units and 204.98 Units of activity. There
33
was also a relatively low amount of protein recovered in either of the two fractions. This
34
was likely due to the majority of the eluted protein containing LDH activity.
35
After the elution, fraction 10 and 11 contained the highest LDH activity with the
36
lowest total protein of all the fractions. These fractions were pooled and assayed as one to
37
compare with previous purification steps.
38
The pooled affinity purified fraction contained 5.4±0.2mg of protein. The fraction was
39
then assayed for enzymatic activity and found to have a relative activity of 2910 ± 20. This result
40
showed a 51±1% total recover of LDH when compared to the original crude lysate. The specific
41
activity of this fraction was 540 ± 20 showing a 19±1 fold change in purity vs the lysate. Taken
42
together, these data show that the affinity purification step was the single greatest fold change in
43
purity when comparing each purification step. Figure 2 clearly shows that LDH bound to the
44
column during both the binding and wash of the column, with only contaminating proteins
45
eluting during the binding stage. Additionally, the elution stage was successful in stripping LDH
46
off of the column, with minimal non-specific proteins eluting.
47
48
Discussion/Conclusion:
49
A two-step Ammonium sulfate salting out procedure first separated the more
50
hydrophobic membranes and organelles, from highly soluble cytoplasmic proteins. The 40% step
51
was not intended to increase the purity of our enzyme, but remove potentially hydrophobic
52
molecules that could clog the affinity column, which was unable to be seen in table 1. Though
53
there was no increase in purity of LDH vs contaminating protein, visually on inspection the
54
homogenate was clear of other cellular debris. The 60% step was intended to separate soluble
55
proteins based on their hydrophobicity. Since Ammonium Sulfate is a highly soluble salt which
56
prefers to bind water molecules preferentially, increasing the concentration will cause other
57
molecules in solution to precipitate based on their affinity for water. At 60% ammonium sulfate
58
we see that LDH a highly soluble molecule can no longer stay in solution. This is shown by the
59
LDH activity of the recovered pellet. Our data shows that there must be more soluble proteins
60
remaining in solution even at 60% ammonium sulfate levels, which is shown by the 2 fold
61
increase of purity after the purification. Comparing the data recovered from the first two
62
purification steps, it is clear the 60% salting out step is superior to the 40% step increasing LDH
63
purity.
64
The affinity purification step was designed to purify out lactate dehydrogenase using
65
AMP-agarose beads and a NAD-pyruvate adduct. The previous steps were designed to remove
66
large hydrophobic molecules and highly soluble ones, but failed to separate out similarly soluble
67
proteins. The agarose beads have a bound AMP molecule that will be used to bind the active site
68
of the LDH molecule and temporarily pull the LDH out of solution and slow down its movement
69
through the solution. Proteins that do not have an affinity for the AMP will not get retarded
70
within the solution and will elute first before any AMP binding proteins. Because there is a
71
limited amount of AMP binding sites if those binding sites were completely saturated then any
72
excess proteins will wash through with the load and rinse fractions. This occurred during our
73
affinity column assay as is shown in Figure 2. During the load fractions there was LDH enzyme
74
present in those fractions, as was shown by qualitative analysis of our spot plate. The LDH in the
75
load fraction was most likely due to over saturation of the AMP- agarose beads leading to the
76
unbound LDH running through the column quickly with all the other non-AMP binding proteins.
77
The affinity chromatography step was the most effective step for increasing the purity of
78
our enzyme. It resulted in an increased of purity, shown by TABLE 1, of 19±1 from the crude,
79
and a yield of 51±1% from our total LDH. This step resulted in a greater increase in purity, and
80
was more effective than previous salting out steps, leading to very little loss of the LDH enzyme.
81
In order to better improve these techniques in the future, replacing the salting out step
82
with a differential centrifugation scheme could lead to removal of hydrophobic molecules and
83
prevent loss of target proteins. Additionally, by performing gel filtration chromatography after
84
the affinity chromatography step, other non-specific binding proteins could be removed based on
85
size.
Figure 1. Hydrophobicity plot of the LDH primary sequence generated from expasy.
TABLE 1
Fraction
Crude (Whole
homogenate)
40% Sup
60% Pellet
Affinity
Chromatography
Total
Protein
(mg)
Relative
Activity
(units)
Relative
Specific
Activity
(units/mg)
LDH
protein
Recovery
(%)
197 ± 4
172 ± 6
63 ± 2
5710 ± 60
4920 ± 20
3590 ± 60
29.0 ± 0.9
29 ± 1
57 ± 2
100
86 ± 1
63 ± 2
1
0.99 ± 0.07
2.0 ± 0.1
5.4±0.2
2910 ± 20
540 ± 20
51±1
19±1
*The data from this table was used to construct Figure 2. *
Fraction Number
1
Fold
Purification
(overall)
Protein Concentration
(mg/ml)
1.788
Enzyme concentration
(units/ml)
0
2
4.747
119.61417
3
5.377
239.22833
4
0.165
119.61417
5
0.039
0
6
0.026
0
7
0.008
0
8
0.010
0
9
0.008
3.497
10
2.256
1321.543
11
0.59
204.98
12
0.079
31.061
13
0.015
3.014
Note on fractions: Load =1-4, Rinse = 5-7, Elute = 8-13
Affinity Column Elution Profile for LDH
7
1400
6
1200
5
1000
4
800
3
600
2
400
1
200
0
Enzyme Concentration (units/ml)
Protein Concentration (mg/ml)
|——-Load——-|——-Rinse——-|————Elute—————|
0
0
1
2
3
4
5
6
7
8
9
Fraction Number
10
11
12
13
14
Protein Concentration
(mg/ml)
Enzyme concentration
(units/ml)
Figure 2: This figure shows the enzyme activity and protein concentration in each elution
fraction. For the load and rinse fractions we used quantitative spot plate results in order to
determine presence of LDH in the fractions. The results were compared to a positive control that
was given an arbitrary (+++) reading and a negative control given a (-). The fractions with a (+)
yielded 1/3 the total activity of the peak (+++) fraction and a (++) yielded 2/3 of the total activity.

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