Hello Sushil,

interesting problem. I don't have an idea what happened but find this: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/flmasbul.Par.0001.File.tmp/flmasbul.pdf there you have a manual for using Anti-Flag Westernblot,maybe you can change conditions for Western blotting.

I agree with an earlier comment that the transfection time may not be enough to produce enough protein to be able to see on the gel. Try longer eg 48 or 72h then test. It is possible that due to the structure of the molecule, expression may not be as high as you were expecting.

I suggest to use target protein specific monoclonal antibody to confirm this result. Also I agree to barbara's suggestion on cultrue time after transfection. In fact, I have ever culture the cell 6 days after transfection. This may not be really good for you. but in my exp, it showed better results than 2 days or 3 days.

Thanks

In addition to the helpful suggestions already provided, I would emphasize that you should run a positive control for transfection (especially if you don't have a lot of experience with the method). Making sure your 293s are growing well and at an appropriate density for transfection will have a major impact on your results, and depending on the methodology used, you may need to transfect in antibiotic-free medium.

Western blots for flag-tagged proteins are generally quite easy. However, if your protein is very small (150kDa), you may miss it due to over or under-transfer. Moreover, while the expectation is that most proteins are recovered in standard lysis conditions, this isn't always the case. Some proteins are secreted, and others may be highly insoluble, and may be difficult to extract. Can you add some details about the expected molecular weights of your cDNAs, and whether anything is known about their cellular localization?

Its really difficult to understand where your problem might be without the sequence reads of your vectors and the inserts: codon usage, protease sites, protein size, hydrophobic content, the list goes on regarding possible trouble points. The answers above should give you some ideas.

First of all thank you so much, I am very happy to get very good suggestion from you all.

As per Dr. Arjan's suggestion, I may not detect the protein because of possibly proteolytic cleavage so I am planning to perform semi quantitative RT-PCR to check expression.

As well as I will increase incubation time for western blot experiments.

I will apply your suggestions and get back to you after a week.

My colleague suggest me to do cloning these genes in another vector, what is your opinion??

I have clone one gene in one vector, but failed to check any expression of this target gene after transfection into 293T cells. However, another 2-3 vectors after insertion of the same gene can express it very well. Until now, I am not sure the real reason for this. in your case, if the sequencing of target genes and strategy for construction are no problem, I still suggest to use target gene specific antibodies (Polyclonal antibody may increase the signal). I have ever used Fugene HD for transfection, which looks better than lipofectamine 2000, Although I never compare them in same experiment. At least the fugene HD do not hurt the cells so much. if you do not clearly know your target gene are secreted or not, you may check both cell lysate and your supernatant. you can use Amicon tube to concentrate the supernatant before adding SDS buffer. Good Luck!