Any suggestions on how to change our protocol to ensure we are excising the right band of Atg4?
We have been attempting purification of the poly his tagged autophagy Atg4 caspase transformed into TOP10 E. coli (ARA and AMP) using FPLC nickel columns. Based on SDS-PAGE and Western blotting with an Atg4 specific antibody, we have successfully purified Atg4 protein in one clean peak. However, everytime we excise the indicated band on our SDS-PAGE in relation to our western blot results (ran on the same parameters at the same time) our sequencing results (sent off to a professional) come back as a significant probability it is protein from E. coli. Any suggestions about changes in the way we are going about this would be greatly appreciated.
Chandramouli Batthula
You must change the transformation protocol and will see the results ....
Paola Di Bonito
Oscar has made goal!
Now I remember ...
If you load your sample in a gel with a big well after blotting you can cut and process by w/b only one stripe of the membrane
Oscar Henrique Pereira Ramos
For direct N-terminal sequencing use PVDF membrane and cut the red ponceau stained band corresponding to the band that is immunorecognized.
Vaibhav Mhaindarkar
do check for its caspase activity..or else you can do 2d gel assay..one on the base of molecular weight and other on the basis of isoelectric point.
and then again try to blot. It will remove probable overlapping of histidine containing bacterial protein and your histidine tagged protein..
Paola Di Bonito
Don' t you can cut the band from the nitrocellulose paper after blotting? Should be some protocol for peptide sequences ...