Two proteins A and B were run on two separate 12% polyacrylamide gels (pH-8.8) i

Question

Two proteins A and B were run on two separate 12% polyacrylamide gels (pH-8.8) in the presence or the absence of SDS. In the absence of SDS, the two proteins migrate at the same position in the gel. However, in the presence of SDS, protein A migrates further than protein B. On a molecular exclusion column, protein B elutes first. Explain these observations. Which protein would you expect to show a higher affinity for a DEAE cellulose resin? Why? Explain your answer and show all your work (3 points)

Explanation / Answer

SDS is an anionic detergent that disrupts a protein native structure and imparts net negative charge on the protein. Two proteins can migrate at the same rate in native PAGE if their charge differences compensate for the size difference. The overall 3d strstructure of the protein i.e. compact or expanded conformation also determines the migration rate of a protein.

In size exclusion column, molecules with greater size are eluted first.

In the size exclusion column, protein with small size enters into the resin while the larger proteins escape through interstitial spaces.

In the given case, protein B is eluted first. So, protein A must show high affinity for the column.

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