Multiple bands in WB - (Jun/28/2007 )
Hello everyone,
I transfected a V5-tagged gene into HEK293, and detected it by western blot. The gene encode a ER enzyme. Beside the expected band, there were many other bands. I think those are not non-specific, since there was no band in mock transfection lane.
Some reasons may account for that:
1. Protein degradation during sample preparation.
2. Proteolytic cleavage in the cell.
3. Internal AUG were recognized as start codon.
Which one is most probable?
-WHR-
QUOTE (WHR @ Jun 28 2007, 10:16 AM)
Hello everyone,
I transfected a V5-tagged gene into HEK293, and detected it by western blot. The gene encode a ER enzyme. Beside the expected band, there were many other bands. I think those are not non-specific, since there was no band in mock transfection lane.
Some reasons may account for that:
1. Protein degradation during sample preparation.
2. Proteolytic cleavage in the cell.
3. Internal AUG were recognized as start codon.
Which one is most probable?
I transfected a V5-tagged gene into HEK293, and detected it by western blot. The gene encode a ER enzyme. Beside the expected band, there were many other bands. I think those are not non-specific, since there was no band in mock transfection lane.
Some reasons may account for that:
1. Protein degradation during sample preparation.
2. Proteolytic cleavage in the cell.
3. Internal AUG were recognized as start codon.
Which one is most probable?
it is to decide by experimental approaches: proteolysis during lysis may abolished by addition of proteases inhibitors; intracellular degradation by blocking of lysosomal/endosomal H+-ATPase acidification (f.i. with Bafilomycin)
-The Bearer-
Thank you Bearer,
I am also interested in the third possibility. Many mRNA contain several AUG in their 5'UTR. However, we usually clone only the coding region for over-expression. How does a cell know at which AUG to start translation?
-WHR-