2 replies to this topic

[SIRHC]

Hi All,

Is anyone working on CBP or at least trying to express CBP in mammalian cell lines?

I am able to clone, PCR, sequence my CBP (CREB Binding Protein) construct but when I transfect it, there doesn't appear to be much detectable expression.

I use a 10% SDS-PAGE gel for analysis...

Any ideas, please? Would appreciate your experiences, advice, etc.

Cheers!
cHRIS

[SIRHC]

Reiterate help needed  

[SaraKay]

Have you checked for expression using inVitro coupled Transcription/Translation  (TNT kits from Promega and others)? This can tell you whether your construct is all correct if you do get a product of the right size.  Once you are sure of that, have you a GFP positive control vector (or LacZ) to check that your transfection efficiency is enough to allow you to detect the protein?
Then, expression in mammalian cells might be low and even if you run a bucketful of lysate on a western, you might not be able to detect it if your antibody affinity is low.  Try immunoprecipitating it from a number of transfected plates of cells pooled 24, 48 hrs post-transfection.  Finally, what cell lines have you tried? Some may transfect better than others, depending on your method of transfection. HEK293 cells work very well with Lipofectamine 2000 for example.
Good luck.