how do you say this ladder like appearance is caused by the gel(did you check some known,control proteins in parallel with the same gel)? are you using ready made gels, if yes try to make your own gels and check it.

it looks to me something going wrong with your antibody or protease inhibitors (I hope it is there in your lysis buffer).

Good luck!

SDS-PAGE is the first approach when you want to detect the presence of proteins in your purification strategy or analyze his conformational structure reducing or denaturing his native structure so it is inevitable maintain the protein integrity performing SDS-PAGE. However you should try NON DENATURING SDS-PAGE adding 0.02% Beta mercapto ethanol (without DTT) and performing the separation at 4-8 C.

Luck!

I am using pre-cast gels as well as self made ones, but until now i haven't tried non denaturing ones. So i might try the latter ones. I can observe this protein appearance on several constructs but definitely not on all proteins. The protein preparation should be fine, i guess. Protease inhibitors are added freshly in the concentrations as it is described in the manufacturer's instructions.

I would try using diluted amount(1/10,1/100) of the same proteins in denaturing gels (I trust more self made, may be I make fresh!). I have a concern with antibody, so I would take a new aliquot and incubate ON at 4deg and 1hr at RT (separately) to check the Ab behavior is fine. I am sure you have heated your sample with a denaturing agent(2-ME or DTT) at 95deg for 5min before loading on the gel.

Also develop a house keeping/control protein in parallel really helps!

Good luck!