Question

3. EGFR (epidermal growth factor receptors) is highly associated with malignant breast cancer. You have generated an antibody against EGFR to screen the breast tissues. However, there are too many proteins in the sera. How do you isolate the antibody against EGFR? What chromatography would you use? What would be coated in the column (solid matrix)?

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Answer 1

The antibody of interest in the sera can be isolated by subjecting the serum to a purification process. Protein of interest can be purified from a crude sample by chromatographic techniques. In this case, affinity chromatography can be used. Affinity chromatography is utilizes specific interactions of proteins. Eg: interaction between antigen and antibody , enzyme and substrate etc.

The stationary phase consist of an inert solid matrix (usually agarose). Here, the solid matrix has be coated with EGFR. When the sera containing the antibody against EGFR is passed through the column, binding of EGFR and antibody occurs. Unwanted proteins leave the column with wash buffer. The bound protein can isolated by changing the pH and/or ionic composition of the buffer.