Question

In: Biology

A graduate student just finished running a western blot and was shocked to see that his...

  1. A graduate student just finished running a western blot and was shocked to see that his protein of interest was not present in any of his samples. Prior to running the western blot, he saw a band at the right molecular weight using SDS-PAGE analysis. Using your understanding of western blotting and SDS-PAGE analysis, explain these seemingly conflicting results.

Solutions

Expert Solution

Solution:-

SDS PAGE

  • It is an electrophoresis method for the separation of protein by their mass.
  • Uses Sodium dodecyl sulfate (SDS) and Polyacrylamide gel.
  • Smaller protein molecules travel farther than larger protein molecule.

Western Blotting

  • It is used for detecting specific protein in a sample of tissue extract.

Steps in western blotting

  1. Isolation and extraction of protein from cell
  2. SDS-PAGE
  3. Transfer of proteins from SDS- Page to Nitrocellulose membrane.
  4. Probing and hybridization ( by using specific antibodies against Specific proteins)

Now, we can see that the SDS-PAGE shows a band of proteins during analysis. But after western blotting, we couldn't spot protein of interest.

This is probably because the  transfer of proteins from SDS-PAGE to nitrocellulose membrane haven't occur correctly.

Probable reasons

  1. Larger proteins transfer slowly than small ones. Increasing transfer time might overcome this issue. 2hour blotting will be fine for larger proteins
  2. Transfer from gel to cellulose membrane under an optimum voltage (40V). So, that gel doesn't get become hot. Use ice block for cooling.
  3. Use proper transfer buffer's, eg:- Dunn's transfer buffer.
  4. Tank blotters are more effective than semi-dry blotters.


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