Question

4.     (Challenge) You are purifying a protein from the brain of patients with an unusual type of dementia triggered by a coronavirus and you hypothesize that this protein may be a signature molecule of this type of dementia. To pursue this hypothesis, you collect brain tissue from patients post mortem, do differential centrifugation, density gradient centrifugation, CM (carboxymethyl cellulose) ion exchange chromatography and gel filtration. You then assess the purity of your protein using both native and SDS gel electrophoresis. The following questions relate to this purification series.

a.     You find that after the CM ion exchange chromatography step your protein is not present in the fractions. What is the most likely reason for this result and how might a switch to a DEAE ion exchange column address this problem?

b.    The next purification step uses gel filtration which is designed to further enrich the fraction containing your protein. What quality about the protein that you want to purify is required in order to use this gel filtration column?

c.     Now you plan to assess the purity of the protein using both a native gel electrophoresis and a SDS gel electrophoresis system. You find that the protein appears as a 400,000 Dalton band on the native gel while on the SDS gel it appears as a 200, 000 Dalton band. What does this imply about the nature of the protein?

d.    You are now able to generate a mAb using this protein as the antigen so that you can perform Western Blots. How can this technique be used in the laboratory to assess protein abundance and why is a transfer paper a critical part of this technique?).

e.     Finally, you have convinced yourself that the mAb has high affinity and specificity as per the Western Blot technique. Now you would like to use it to purify lots of the protein using Protein A immunoprecipitation. How does this technique work?

Answer 1

1. Carboxymethyl cellulose (CM) is a negatively charged resin used to purify positively charged proteins (cationic proteins). If after the CM ion exchange chromatography step your protein is not present in the fractions, it means the protein of interest is negatively charged and hence does not bind to the CM resin.

Diethylaminoethyl cellulose (DEAE) is a positively charged resin which can bind the negatively charged protein and purify it efficiently.

2. Gel filtration is a size based separation technique. Large molecules are eluted first and smaller molecules are eluted last.
3. During SDS-PAGE, disulphide bonds between subunits are broken whereas in native PAGE the subunits remain together.

Since the protein of interest shows a molecular weight of 200000Da in SDS-PAGE and 400000Da in Native PAGE, it can be inferred that the protein of interest has 2 subunits of 200000Da each.

4. During western blotting, the monoclonal antibody (mAB) binds specifically to the protein of interest present on the membrane. This produces a band proportional to the amount of protein present in the sample. By comparing the size of the band to that of known marker, we can calculate the amount of protein present in the sample.

The transfer paper used in western blot is critical. There are 2 types of membrane used, PVDF and nitrocellulose. These membranes have high protein binding capacity. PVDF is generally used for high molecular weight proteins while nitrocellulose is used for low molecular weight proteins.

5. During protein A immunoprecipitation, the sample containing the protein of interest is incubated with the antigen specific mAB we have developed. This antigen-antibody complex is incubated with beads containing protein A which binds to the Fc region of antibody present in the antigen-antibody complex in reversible manner.