Hi Muhammad, if I were you I would use the ChromoTek GFP-Trap for IP, it is very efficient and it works on YFP (see first link below).

For WB the Roche Ab has been widely used on GFP. The differences between EGFP and YFP are minimal (I made an alignment for you in the second link, should be visible for a week), and most Abs that recognize GFP can also recognize YFP, but I don't know for sure for the Roche Ab, it depends on the epitope, since it is a monoclonal. You should have in your WB a GFP sample of some kind (any lysate of cells transfected with a GFP construct) as positive control to be sure your WB conditions are ok. If then the GFP sample is recognized but not your YFP sample, then you know for sure. In that case you can buy one of the Clontech antibodies, for example the Living Colors® A.v. Monoclonal Antibody (JL-8) which they sell also in small aliquot for smaller price and works well at very high dilution (1:5000).

Good luck.

http://www.chromotek.com/products/nano-traps/gfp-trap/

http://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20151015-104844-0998-43312320-pg

Hi Pietroo, Thanks for your suggestion. I am using seedling extracts to test whether it works with YFP or not, will see how it goes otherwise I can use GFP trap. Thanks.

I have actually used the Roche antibody for IP of a YFP fusion protein many times.  I then blot with rabbit anti-GFP from Cell Signaling.  This works well and there is very minimal detection of heavy and light chain from the mouse antibody in the sample.  I don't think either antibody is affected by the mutation that makes GFP into YFP.  My experience is with mammalian cells however.  Good luck.

Hello friends, could someone give me some suggestions.

I am using Roche anti-GFP antibody (Cat#11814460001) for detection of my gene of interest in Hela cells using western blotting. My secondary antibody is antimouse IgG HRP conjugated

This antibody successfully detects the EGFP but doesn't detect my gene of interest.

I use lipofectamine 3000 with opti medium and usually exchange medium to 2% FCS MEM after 24 HRS.

Could this failure be attributed to the failure of Epitope recognition or what is exactly the problem?

Thank you in advance.