Protein Lab Report

Fariha Omar, Molly Leary, Annette Crowley

Bio L111

Department of Biology

Suffolk University

Boston, MA 02114

11 April 2017

RESULTS AND DISCUSSION

The purpose of this protein module experiment (from weeks 5-8) was to analyze the protein composition

of IgG antibodies.

WEEK 5:

We received a sample of rabbit blood which we centrifuged to separate the plasma from the rest of the

cells. We used the BCA Assay to create a standard curve representing the average absorbance of the

duplicate samples of the BSA standards against the amount of protein in each of the tubes (Figure 1). The

equation generated by the standard curve was used to determine the protein concentration of the plasma

samples (Figure 2, column 3). And then we multiplied the dilution factors to get the concentration of the

original samples (Figure 2, column 4). We saved two tubes of plasma samples for the following weeks.

WEEK 6:

We used a technique known as affinity purification to purify the IgG antibodies from one of the

untouched plasma sample from week 5. Because Protein A binds specifically to IgG antibodies, we mixed

Protein A beads with the plasma sample to help us isolate the IgG antibodies. In order to separate the IgG
antibodies from the rest of the proteins in the plasma sample, we centrifuged the tube which pulled the

Protein A and IgG antibodies to the bottom of the tube, then we washed the beads with loading buffer to

get rid of the unwanted proteins. We removed the supernatant and saved it in another tube and labeled it

flow-through. (The flow-through tube should contain the protein components of plasma other than the

IgG antibodies.) Lastly, we removed the IgG antibodies from the beads by adding a low pH elution

buffer. We labeled this purified antibody. (The purified antibody tube should only have IgG antibodies).

WEEKS 7 & 8:

On week 7, we used SDS polyacrylamide gel electrophoresis (SDS-Page) to analyze the untouched

plasma sample (week 5), flow-through sample (week 6), and purified antibody sample (week 6).

Electrophoresis uses electricity in a gel polymer to separate similar proteins based on their mass

properties.

On week 8, we received a picture (Figure 3B) of our SDS-PAGE gels (from week 7) in order to analyze

the successfulness of our IgG purification and to examine the subunits that exist in an IgG antibody. The

lanes are labeled based on the three (plasma, flow-through, and purified antibody) samples.

According to Figure 3B, there are more bands in the plasma sample lane than there are in the flow-

through. The reason for this is the plasma sample is unpurified. It should contain all the proteins,

including IgG antibodies. Whereas, the flow-through sample is the supernatant from the affinity

purification centrifugation process, in which IgG antibodies were removed, thus it should contain proteins

other than the IgG antibodies. However, through comparative observation of the other two lanes, we

concluded that the bands in the flow-through sample lane are antibody proteins.

The IgG antibody is made up of two heavy chains (of the molecular weight 50kD) and two light chains

(25kD) that are held together by disulfide bridges. When electrophoresis was applied to the antibody (in

week 7), the three dimensional structure of the antibody was denatured and the bonds were broken, letting

the chains loose and separate based on their mass in the gel. In the purified antibody lane, the bands
represent the chains of an IgG antibody. In figure 3B, the bands align with the weight standard (figure

3A) proving that the bands within the gel are the heavy and light chains of the IgG antibody.

In addition to the plasma, flow-through, and purified antibody samples, the leftmost lane of Figure 3B

presents a weight marker of known molecular weights that was also loaded into the gel. Using that

marker, we calculated each bands migration distance (Figure 4, column 3) in centimeters, then found the

relative mobility (Rf values) by dividing by the furthest band (Figure 4, column 4). We compared it

against the log molecular mass (Figure 4, column 2). Plotting the Rf values on the x-axis against the log

molecular mass values on the y-axis, we generated a calibration curve (Figure 5) with which we found the

linear equation to find the molecular weight values the bands of our plasma and purified antibody samples

(Figure 6 & 7).

FIGURES:

Figure 1: BSA Assay Standard Curve

Figure 1: BCA Assay Standard Curve generated an equation which we used to find the unknown protein
concentration of plasma sample.

Figure 2: Plasma Sample Concentration

Figure 2: This table shows the protein concentrations of the diluted and original plasma samples.

Figure 3: Protein Gel

FIGURE
3A

FIGURE 3B.

Figure 3: Picture of the stained SDS-PAGE for IgG antibody analysis is shown on FIGURE 3B. Weight
standard that was used to create molecular weight marker is pictured in FIGURE 3A, the middle three lanes
(FIGURE 3B) represents our plasma, flow-through, and antibody samples.

Figure 4: Molecular Weight Markers

Figure 4 shows the calculations of the gel bands of the 3 samples, from Figure 3, to find Rf values and log
molecular mass values.

Figure 5: Standard Curve Molecular Weight

Figure 5 shows the linear equation of standard curve that was generated by the log of molecular weight
plotted on the y-axis, against the Rf values plotted on the x-axis.
Figure 6: Molecular Weight

Figure 6: Using the equation generated by the standard curve in figure 5, protein sizes were calculated for the
plasma sample.

Figure 7: Molecular Weight

Figure 7: Using the equation generated by the standard curve in figure 5, protein sizes were calculated for the
antibody sample.
CONCLUSION

The resulting week of this experiment proves that our experiment did not go as flawlessly as we may have

wanted it to. The lanes on Figure 3 show an almost identical number of bands. This should not have been

the case. The flow-through sample lane should not have had the same amount of bands as the purified

antibody sample lane.

In week 6, when we created the flow-through sample, we had put in Protein A beads to capture all the

antibodies so that the supernatant (flow-through sample) after centrifugation had been free of antibodies. I

believe that there were not enough beads to capture all the IgG antibody molecules in the original rabbit

plasma sample. Due to the shortcomings, we were unable to completely compare samples containing IgG

antibody molecules against a sample without.