Affinity Chromatography

Affinity Chromatography

Purpose

This laboratory session aimed to perform the purification of IgG using a Protein A column and estimate its molecular weight using SDS-PAGE.

Introduction

Affinity chromatography is a separation method that is commonly applied in biochemistry. Mainly, as explained by Bahadir, Saltan, Ozbilgin, and Ergene (2013), it is used for the separation of biochemical mixtures based on a highly specific interaction that happens between antigens and antibodies, enzymes, as well as substrates, receptors, and ligands. The interactions can also be with proteins and nucleic acids. Hage et al. (2012) postulate that this is a chromatographic separation technique that is used in the laboratory mainly for the purification of biological molecules in a mixture by basing on their different molecular properties. For instance, the elution of proteins by the use of a ligand solution can be employed in this process.

Biological molecules, including enzymes, and proteins, can freely interact with other molecules, specifically by the use of different bonds. Hydrogen bonding, ionic interaction, disulfide bridges, and hydrophobic interactions are among the types of interactions and bands that are commonly observed (Hage et al., 2012). The specificity is a consequence of desired molecules being allowed to interact with the stationary phase and to bind inside the column. By this selective biding, one component of the mixture can be separated from the rest of rhe mixture. The use of an elution solvent does the elution of the bound components. This causes competitive interaction between the molecules and the stationary phase, causing the elution of the bound molecules. A change in pH or a change in ionic strength may also be used to facilitate elution.

Affinity chromatography, based on the fact that it is a highly promising, attracts significant application. Primarily, it separates proteins based on their interaction with specific ligands. The ligand, in this case, is coupled with the chromatography matrix. The technique hence is useful for purification of proteins when the ligand is known. When used for separation, it presents a high selectivity, resolution, and capacity for the protein of interest. Alongside this, it also resents a thousand-fold purification capability with a high rate of protein recovery. The target protein is obtained in a purified and concentrated form.

Three main stages are involved in the process. Firstly, the affinity medium is equilibrated in a binding buffer. Secondly, adsorption of the targetted molecules is performed, and the unbound material eluted. The elution of the target molecules is the third stage. The fourth stage involves the creation of a re-equilibrium.

SDS-PAGE makes up the second part of this experiment. It is a reliable method of approximating molecular weights for proteins of unknown name. In the SDS medium, the migration of proteins is based on molecular weight. Specifically, the movement is in an inverse relationship with the molecular weight (Nowakowski, Wobig & Petering, 2014). This means that heavy molecules move the least while the lighter ones move them further. Thus, by carrying out electrophoresis in SDS PAGE, it is possible to approximate the molecular weights of the eluates.

In the current experiment, the column matrix is covalently linked to protein A. the protein is obtained from Staphylococcus aureus and binds IgG molecules from different species. It is used, therefore, in the purification of proteins by changing pH and ionic strength. The strength of the bond depends on the species from which the antibody is obtained. After the separation stage, the eluates are run through SDS-PAGE for the estimation of molecular weight.

Materials

1. Tris buffer pH 8.0
2. Chromatography column
3. Serum sample
4. 5M Sodium chloride
5. 3M magnesium chloride
6. Phosphate buffered saline
7. SDS-PAGE gel

Method

1. Purification process
2. Equilibrate protein A column with 7.5 ml of 50 mM Tris, pH 8.0.
3. Dilute 5 mL serum with 5 mL of equilibration buffer.
4. Run diluted serum over equilibrated column 2X, retain the “flow-thru” after the second time as a back-up.
5. Wash column 2X with 7.5 mL of 50 mM Tris (pH 8.0), 0.5 M NaCl.
6. Elute with 10 mL of 3 M MgCl2. Transfer eluate to dialysis bag.
7. Dialyze overnight in 1 L PBS (pH 7.4). The following day change the buffer two more times
8. To strip column for reuse with a new sample, run another 7.5 mL of 3 M MgCl2 through column and re-equilibrate before loading the next sample.
9. If the same sample is to be used, it is only necessary to re-equilibrate between loadings.
10. After use, run 7.5 mL of 20-% ethanol through the column and then store the column in this solution at 4oC
11. Analysis of the purified product
12. Run 12 µL of each sample in SDS-PAGE gel [SDS-PAGE protocol]:
13. Ladder
14. Serum (control)

• Flow-thru

1. Wash 1
2. Wash 2
3. Eluate

• Stripped column eluate

Results

After running the electrophoresis, the results appeared as shown below in figure 2

Figure 2: SDS-PAGE results. The image presents the results obtained after carrying out the electrophoresis of the eluted sample. As can be seen, the electrophoresis resulted in distinct bands of coloration is an indicator that the sample used contained different constituents. The single bright bands define the product of interest. Ton comparing with the control serum, it is confirmed that the eluates include the IgG.

Analysis and Discussion

SDS PAGE separates proteins based on their molecular weights. Proteins can be denatured (boiled to isolate a protein complex into monomers). β-Mercapto-ethanol is then added to break disulfide bonds (Zhang, 2018). SDS, on the other hand, is a detergent that makes proteins negatively charged. SDS-PAGE gels are commercially produced and come in different gel percentages (for different pore sizes). SDS-PAGE separates based on molecular weight. Thus, analyzing this method leads to an approximate molecular weight, which is obtained by comparing it with the molecular ladder used.

When analyzing an SDS PAGE result, it is vital to observe for reproducible patterns of the bands. This can be concluded by comparing it with the molecular ladder used. Precisely, it is unlikely that two unrelated compounds can yield the same patterns unless they have the same molecular weight. Therefore, herein, the IgG is separated from the rest of the eluate by the difference in molecular weight. Notably, the samples contain one band in stock, as seen in the photo above. This confirms the presence of IgG.

Conclusion and Application

The experiment aimed at carrying out the purification of IgG using a protein A column by way of affinity chromatography. Following the purification, the molecular weight was approximated using SDS-PAGE. Proteins are amphoteric. Their net charge, therefore, is always determined by the pH of the solution ib which they are suspended. When pH is above the isoelectric point, the charge on the protein surface is negative. When it is positive, the charge is positive. Whereas tye charge on proteins differs depending on the medium, that of nucleic acids always remains negative. Thus, the separation of nucleic acids happens only based on size. These are among the applications of SDS-PAGE.

References

Bahadir, O., Saltan, G., Ozbilgin, S., & Ergene, B. (2013). Affinity Chromatography and Importance in Drug Discovery. Column Chromatography. doi: 10.5772/55781

Hage, D., Anguizola, J., Bi, C., Li, R., Matsuda, R., & Papastavros, E. et al. (2012). Pharmaceutical and biomedical applications of affinity chromatography: Recent trends and developments. Journal Of Pharmaceutical And Biomedical Analysis, 69, 93-105. doi: 10.1016/j.jpba.2012.01.004

Nowakowski, A., Wobig, W., & Petering, D. (2014). Native SDS-PAGE: high-resolution electrophoretic separation of proteins with retention of native properties, including bound metal ions. Metallomics, 6(5), 1068-1078. doi: 10.1039/c4mt00033a

Zhang, Y. (2018). SDS-PAGE for Silk Fibroin Protein. BIO-PROTOCOL, 8(20). doi: 10.21769/bioprotoc.3054