Lab Report 6: Microplate Protein Quantification, Sample Normalization, and Purification
Table
Objective: To determine the success of the affinity and IEX chromatography purifications
through calculating the percent yield and fold purification of GST-GFP and to equalize the
protein concentration of each of the samples through sample normalization.
Principle of Methods: In order to determine the percent yield and fold purification of GST-GFP
from the original lysate, affinity and IEX purification, protein concentrations must first be
determined. Protein concentrations from each of these samples can be determined through
utilizing the DC assay. The DC assay will be performed using a microplate and plate reader in
order to increase the efficiency and precision of the assay. A standard curve will be generated
and used to determine the protein concentrations of each sample. Sample normalization will also
be performed in order to ensure that each of the samples have equal amounts of protein,
therefore, allowing for more accurate analysis in things such as SDS-PAGE. A purification table
will then be used to determine the success of each of the purification steps, performed in labs 4
and 5.
The DC protein assay was performed using a microplate and plate reader. The DC assay is
performed in the same manner as described in lab 2, however, instead of using individual
cuvettes, a microplate reader is used. Loading individual cuvettes into the spectrophotometer is
time consuming and leaves more room for error. Utilizing a microplate and plate reader speeds
up the process and increases the precision of the assay. The microplate has 96 wells, therefore,
allowing for 96 samples to be loaded and analyzed at the same time. The microplate reader is a
machine that can individually read each of the absorbances of the microplate wells. It passes
light vertically through the sample and measures its absorbance using a spectrophotometer. Due
to the fact that the light is passed vertically through the sample, as opposed to horizontally when
using individual cuvettes, it is very important that the volume of the samples are consistent.
Inaccuracies in volume will cause the absorbance to be read inaccurately. Using the microplate
and plate reader is simple. Just prepare the DC assay as indicated in lab 2 and load the samples
into the microplate reader. Then insert the microplate into the plate reader, set the absorbance to
750nm, and press “read.” The machine will automatically read each of the individual wells on
the plate and record each of the corresponding absorbance values on a computer.
Sample normalization is performed in order to ensure that each of the protein sample are of equal
volume. Due to the fact that each of the purification steps results in the loss of some protein, it is
known that the proteins will be of difference concentrations within each of the samples. Equal
protein concentrations between the original lysate, affinity chromatography, and IEX
chromatography samples is important for accurate analysis during tests such as SDS-PAGE.
Equal volumes of total protein will ensure that the purification of the protein of interest can be
analyzed correctly. Sample normalization is performed by simply multiplying the protein
concentration (found by the standard curve generated from the DC assay) of the sample with the
lowest value, not including flowthroughs, by the total volume of sample needed for the
upcoming experiments. This number is then divided by each of the individual samples’ protein
concentrations to determine the volume needed of each sample to obtain equal concentrations.
These sample volumes are then subtracted from the total volume of sample needed in order to
find the amount of water to add to each of the samples. Following this procedure will result in
samples of needed volumes with the same concentration of total protein in each.
A purification table is very useful in quantitatively determining the success of individual
purification steps. A purification table will be used to determine the purity of GST-GFT of the
affinity and IEX chromatography compared to that of the original lysate, therefore, determining
the success of each of these steps. The purification table will be used to determine the total
activity, specific activity, percent yield, and fold purification of GST-GFP using the data from
the previous labs. The purification table is completed through a series of calculations. Total
protein concentration and total activity of GST-GFP are expected to decrease throughout the
purification steps due to the nature of the chromatography steps in losing some of the protein
through the process. They are determined through the following equations:
 protein concentration (mg/ml) x total volume (ml) = total protein (mg)
 reaction velocity (units/ml) x total volume (ml) = total activity (units)
Percent yield measures the amount of GST-GFP retained in each step of the purification process
compared to that of the total protein. Both total percent yield and percent yield of GST-GFP are
expected to decrease throughout the purification because it is expected that some protein will be
lost with each step. Total percent yield and the percent yield of GST-GFP are calculated by the
following equations:
 percent yield total = total protein of sample/total protein of lysate x 100
 percent yield of GST-GFP = total activity of sample/total activity of lysate x 100
Specific activity is another way of determining the success of the purification steps. Comparing
the change in specific activity to the change in total activity is a good way of quantifying the
purity of each of the samples and therefore, determining the success of each of the purification
steps. Total activity is expected to decrease because of the loss of protein with each purification
step. In contrast, specific activity is expected to increase as the purity of GST-GFP increases
throughout the purification process. They can be calculated using the following equations:
 total activity (units) = total volume (ml) x reaction velocity (units/ml)
 specific activity (units/mg) = reaction velocity (units/ml)/protein concentration (mg/ml)
Fold purification quantifies the increase in the purity of the target protein, GST-GFP. Increase in
fold purification with each of the purification steps indicates that the target protein was
successfully further purified. The fold purification can be determined by the equation:
 fold purification = specific activity(of column)/specific activity (of original lysate)
Materials:
 Reagent A - Alkaline copper titrate.
 Reagent B - Folin reagent.
 Reagent S – 10% Sodium dodecyl sulfate (molecular weight = 288.38).
 E. coli BL21(DE3) lysate containing a GST-GFP fusion gene, transformed with a pET
expression vector.
 96-Well Plate – flat bottom, polypropylene, 96-well plate (purchased from USA
Scientific).
 Plate Reader – Tecan Infinite M200.
 Affinity Flowthrough Sample – Collected in lab 4.
 Affinity Elution Sample – Collected in lab 4.


IEX Flowthrough Sample – Collected in lab 5.
IEX Elution Sample – Collected in lab 5.
Results:
Table 6.1: DC Protein Assay BSA Absorbance Values
Sample #
BSA (mg/ml)
Absorbance
(750nm)
A1
0
0.0768
A2
0
0.0666
B1
0.3
0.0858
B2
0.3
0.1035
C1
0.6
0.1212
C2
0.6
0.1201
D1
0.9
0.133
D2
0.9
0.1547
E1
1.2
0.127 **
E2
1.2
0.1626
F1
1.5
0.1912
F2
1.5
0.2009
G1
1.8
0.2014
G2
1.8
0.2408
**Erroneous data not included in final graph.
Table 6.2: DC Protein Assay Lysate Absorbance Values
Sample #
Absorbance
Amount (mg/ml)
(750nm); Plug in from BSA
as “y”
Standard Curve,
Solve for “x”
A3
0.2786
2.5127
A4
0.2709
2.4198
B3
0.1167
0.560
B4
0.1281
0.697
C3
0.2168
1.767
C4
0.1898
1.441
D3
0.1111
0.492
D4
0.1027
0.391
Average Protein
Concentration
(mg/ml)
2.4663
0.6285
1.604
0.442
E3
0.1105
0.4849
E4
0.1167
0.5597
0.523
Equation 6.1 (Standard Curve) – y = 0.0829x + 0.0703
Value 6.1 (Protein Concentration of Original Lysate) – 2.466 mg/ml
Value 6.2 (Protein Concentration of Affinity Flowthrough) – 0.6285 mg/ml
Value 6.3 (Protein Concentration of Affinity Elution) – 1.604 mg/ml
Value 6.4 (Protein Concentration of IEX Flowthrough) – 0.442 mg/ml
Value 6.5 (Protein Concentration of IEX Elution) – 0.523 mg/ml
Value 6.6 (Total Protein) – 26.15 g
Value 6.7 (Volume of Lysate for Normalization) – 10.604 g
Value 6.8 (Volume of Affinity Flowthrough for Normalization) – 41.607 g
Value 6.9 (Volume of Affinity Elution for Normalization) – 16.303 g
Value 6.10 (Volume of IEX Flowthrough for Normalization) – 59.163 g
Value 6.11 (Volume of IEX Elution for Normalization) – 50.0 g
Table 6.3: Sample Normalization
Sample
Total Protein (g)
Sample Volume (l)
dH2O to Add
26.15 g
10.604 l
39.396 l
Affinity Flowthrough 26.15 g
41.607 l
8.393 l
Affinity Elution
26.15 g
16.303 l
33.697 l
IEX Flowthrough
26.15 g
59.163 l **
0 l
IEX Elution
26.15 g
50.0 l
0 l
Lysate
**50 l added, not 59.163 l.
Table 6.4: Purification Table for GST-GFP
Protein Total
Total
Reaction Total
Specific
Percent Percent
Conc.
Volume Protein Velocity
Activity Activity
Yield
Yield
(mg/ml) (ml)
(mg)
(units/ml) (units)
(units/ml) (total)
(GSTGFP)
Lysate 2.466
5
12.33
1.75
8.75
0.710
100%
100%
Affinity 1.604
4.5
7.218
1.03
4.65
0.642
58.54% 53%
Elution
IEX
0.523
1.0
0.523
1.06
1.06
2.027
4.242% 12%
Elution
Fold
Purif.
(GSTGFP)
1
0.904
2.855
Graph 6.1: Standard Curve (Including Erroneous Data)
Standard Curve
0,3
y = 0,0804x + 0,0695
R² = 0,9156
Absorbance (750nm)
0,25
0,2
0,15
0,1
0,05
0
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
BSA Concentration (mg/ml)
Graph 6.2: Standard Curve (Excluding Erroneous Data)
Standard Curve
0,3
y = 0,0829x + 0,0702
R² = 0,9621
Absorbance (750nm)
0,25
0,2
0,15
0,1
0,05
0
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
BSA Concentration (mg/ml)
Discussion: The purpose of this lab was to utilize a purification table to quantitatively analyze
the results of the affinity and IEX chromatography purifications and to perform sample
normalization to equalize the protein concentrations of each sample. A DC protein assay was
performed using a microplate and plate reader to generate a standard curve and determine the
protein concentrations of each of the samples. Sample normalization was performed, giving each
of the samples equal concentrations of protein for future use. A purification table was
constructed using the values obtaining from this lab as well as previous labs and was used to
quantitatively analyze the success of the purification procedure.
Graph 6.1 shows the standard curve obtained from the BSA absorbance values in table 6.1. The
R^2 value of this graph is 0.9156, indicating sources of error. The primary source of error is due
to an erroneous data point, sample E2, which was likely caused by a pipetting error. When this
erroneous data point is not included in the final graph the results show a much higher level of
precision within the experiment, as shown in graph 6.2. The R^2 value of graph 6.2, excluding
the erroneous data, is 0.9621. As shown by the significant change in R^2 values when comparing
graphs including and excluding the erroneous data, it can be determined that a mistake was made
during pipetting of this sample and therefore, this data point will not be included in the final data.
Using graph 6.2, the standard curve equation is y=0.0829x + 0.0703. This equation was used to
calculate the protein concentration of each of the samples, as shown in table 6.2.
The protein concentrations, calculated using equation 6.1, can be analyzed to provide more
information about the purifications that were performed. The protein concentration of the
original lysate (value 6.1) was the highest at 2.466 mg/ml. The original lysate is expected to
contain the most protein because nothing has been done to this sample to remove any of the
protein. As the name indicated, it is the starting material, therefore, it is expected to contain the
highest concentration of protein. The second highest protein concentration was the affinity
elution at 1.604 mg/ml. The affinity chromatography was the first purification step to be
performed so the protein concentration is expected to be lower than that of the original lysate
because some of the proteins are removed with this step. The affinity flowthrough has a protein
concentration of 0.6285 mg/ml. The flowthrough protein concentration is lower than that of the
elution because the target protein, as well as some contaminant protein, are trapped in the beads
of the affinity column. Therefore, the majority of the protein will not come out in the
flowthrough and will instead be caught in the elution sample. This is the reason why the elution
has a higher concentration than that of the flowthrough. The IEX elution has a protein
concentration of 0.523 mg/ml which is lower than that of the affinity elution. This is due to the
fact that the IEX purification was performed after the affinity. Some of the protein was removed
from the sample during the affinity purification and even more was removed during the IEX,
causing its concentration to be the lowest of the elution samples. The protein concentration of the
IEX flowthrough was 0.442 mg/ml, the lowest of all the values. The concentration of the protein
in this step was lower than that of the IEX elution because of the same reason, explained
previously, that the affinity flowthrough was lower than the elution. Most of the target protein
was retained in the IEX column only allowing some contaminant protein to be washed out in the
flowthrough while the majority was washed out with the elution.
When comparing the protein concentration of the original lysate, calculated during this lab, to
that of the values calculated in lab 2, there are notable differences. The protein concentration
calculated from the DC protein assay in lab 2 was 2.039 mg/ml (value 2.2). The protein
concentration calculated from the Bradford assay in lab 2 was 1.305 mg/ml (value 2.3). The
differences between these two numbers can be explained by the inherent differences in precision
between the two assays. The average of these two values is 1.672 mg/ml (value 2.4). When
comparing the protein concentration of the original lysate, calculated in lab 2, to the number
calculated in lab 6, it is obvious that were inaccuracies present. The protein concentration of the
original lysate calculated in lab 6 is 2.466 mg/ml (value 6.1), much higher than the values in lab
2. This difference could partially be due to the differences in procedures between the two labs.
While both labs did use the DC assay to determine protein concentration, lab 6 utilized a
microplate and plate reader while lab 2 was performed using individual cuvettes. It is known that
a microplate reader is a more precise method and the increased human error present, while
performing the DC assay with the individual cuvettes during lab 2, is a likely reason for the
differences between the two values.
Analyzing the purification table for GST-GFP (table 6.4) is a useful in determining the success
of the each of the purification steps. The protein concentration in table 6.4 shown a decreasing
trend throughout the purification steps. This is an expected result because it is known that some
of the total protein will be lost with each step of the process. Protein is lost through the nature of
the purification process as contaminant proteins are removed. Protein is also lost due to factors
such as degradation. Freezing and thawing a protein, keeping it as room temperature for
extended amount of time, and handling it are all things that can cause the protein to denature,
therefore, decreasing the protein concentration. The total volume in table 6.4 also shows a
decreasing trend. This is another expected result as only small fractions of elutions, with the
highest concentration of the target protein, were kept from each step of the purification process,
therefore, the volume decreased with each step. The total protein showed a decreasing trend for
the same reasons that the protein concentration decreased throughout the purification process.
The reaction velocity in table 6.4 decreases from the original lysate to the affinity elution but
then increases slightly from the affinity to the IEX elution. The reaction velocity measures the
rate of the reaction and therefore, the amount of GST. This explains why there is a decrease
between the original lysate value and the affinity elution value. Due to the nature of the
experiment, it is expected that GST will degrade and lose its activity over time. Freezing and
thawing as well as sitting as room temperature will cause the protein to degrade and therefore,
lose its activity. A decrease in activity will show a decrease in reaction velocity. The slight
increase in reaction velocity between the affinity elution and IEX elution is not expected and is
due to human and experimental errors. However, the minor increase of 0.03 units/ml indicates
that there were likely no major errors involved but rather, error that is normally expected within
an experiment.
Table 6.4 indicates that total activity decreases consistently throughout the purification process.
This is an expected result because the nature of the purification process is to remove contaminant
proteins, it is expected that some target protein will be lost as well, therefore, total activity is
expected to decrease. In comparison to total activity, which measures the activity of all proteins
in the sample and not just the target protein, specific activity measures the activity of just the
target protein, GST-GFP. As GST-GFP becomes more pure with each step of the purification
process, it is expected that the specific activity will increase. Table 6.4 shows a significant
increase in specific activity between the affinity and IEX elutions, indicating that there steps
were very efficient in purifying GST-GFP. However, the specific activity slightly decreased
between the original lysate and the affinity elution. This decrease could be due to that fact that
some GST-GFP activity was lost between these steps. As discussed previously, the nature of the
experiment and the significant amount of time between these two steps likely caused some of the
GST-GFP to degrade and lose some of its activity. The decrease in specific activity between the
original lysate and the affinity elution was only 0.059 units/mg. This minor of a decrease
indicates that there were no major errors in the experiment but that the decrease was just caused
by the expected degradation of the protein.
The total percent yield and percent yield of GST-GFP both show decreasing trends in table 6.4.
These are both expected results as the nature of the purification process is to remove contaminant
protein and some target protein is also expected to be lost during this process. The final percent
yield of the IEX elution show that the GST-GFP has a higher percent yield than that of the total
protein. This is also an expected result because as GST-GFP is purified, more of the
contaminated protein must be lost than the target protein in order for the purification to be
successful. The 12% yield of GST-GFP indicated that 12% GST-GFP was retained from the
original sample throughout the purification process.
The fold purification of GST-GFP shows the “fold” increase of the protein of interest in relation
to the other protein contaminants. The higher the fold purification, the more successful the
purification was in retaining GST-GFP while removing contaminants. Table 6.4 shows that the
fold purification was 2.855 after the IEX chromatography. This increase in fold purification
indicates that the purification process was successful in retaining GST-GFP while removing
contaminants.
Through analysis of the purification table, it can be determined that both steps, the affinity and
IEX chromatography, were useful in removing contaminants while retaining GST-GFP.
Although there were some minor inconsistencies, as discusses previously, between expected and
actual results, they were all small and could all be explained. When comparing the fold
purifications, the IEX purification seems to have been the most beneficial step in the purification
scheme, however, the affinity chromatography was beneficial as a first—step in eliminating
many contaminants before performing the IEX. Therefore, both steps are recommended for
performing GST-GFP purification.
In conclusion, through analysis of the purification table, it can be determined that both affinity
and IEX chromatography are beneficial steps in the purification process of GST-GFP. The
change that would be most beneficial in maximizing the purification of GST-GFP would be to
perform the purification scheme in a shorter time period, minimizing degradation of the target
protein.
Sources: Borgon, R and Nicole Verity. Quantitative Biological Methods. Seventh Edition.
Hayden-McNeil, 2018. Print.