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