Western Transfer and Detection of small protein - (Jan/23/2007 )

Hi,

I recently am running into problem of western detection of a small protein of ~4-5KDa which I am trying to expressed in 293T cells. the peptide sequence is tagged with N-terminal HA from the vector and sequencing results have shown the entire construct to be correct, in frame and proper stop codon.

I tried to lyse the cells collected after transient transfection (previously checked to show high transfection efficiency through eGFP) using tritonX100-NaCL-Tris buffer with complete protease inhibitor. Cells were sonicated for a short while and clarified by spinning at 15,000RPM at 4oC for 15 minutes. This preparation yielded no result.

I tried direct adding of 2X SDS sample buffer to cell pellet but running the sample resulted in very sticky loading. Also, seems that blocking cannot completely work on the higher molecular weight as I have lots of non-specific binding even in untransfected sample.

The gels ran were 15% Tris Tricine and transfer was done using transfer buffer with 10% methanol 21V for 20 minutes using semidry on nitrocellulose.

Recently, I tried PVDF and it didnt work too. Basically, PVDF was soaked for 2 minutes in methanol and then washed 1min x2times with dH2O. After that, membrane was soaked in transfer buffer with 10% methanol until transfer.

I am really thinking hard as to what else is wrong and wonder if anyone can point me to good light on any critical mistake in what I have done.

Thanks a lot in advance.

Cheers,
tltan

There might be a lot of possibilities (in the cell, protein you are working with might be lowly expressed ( load to the SDS gel with high amount of protein 50 or 100) ; antibody might be non-spesific or antibody dilution might not be correct (try low dilution or high dilution); the protein might go out of gel when you are running (look at the marker at similar size) etc

Where is your protein located in the cell? This affects your experiment. Due to low molecular size, the transfer of protein of interest to the membrane might be quick. Try low hours during the transfer.

Good luck

hello its also possible you are semi dry blotting for too long or at too high a voltage.
i regulary blot for proteins of 12-13 kda, and always run at 13V for 15 mins. any longer and i normally lose some of even this size. its possible yours are running through the membrane?
hope this helps

Try using a membrane with a smaller pore size. If your membrane is 0.45 micron, proteins smaller than 20kDa tend to go straight through. You can use 0.2u or there may be even smaller. Otherwise its possible you're not getting expression or the antibody is not working (HA is usually pretty good, but have you tried the Ab on a different HA protein?).

Hi,
Try 0,2uM membrane,it should work, because with bigger pores I assume your protein go away. And another I think your gel concentration is important. I don't know what percentage you should use since I haven't worked with that protein size.Maybe 4-15% tris-Hcl gel works but,I'm not sure but you can find it in the papers. I hope it works!
celtichigh

Hi all,

thanks for your reply.

This vector have been tested to work well. the monoclonal anti-HA antibody have been proven to work on the expressed protein from this vector transfected cells as well.

The insert that I am dealing with is a 60amino acid region of a protein. Sequencing showed that everything is correct and in frame since its N-terminal HA from the vector. I added 2 stop codon at the end.

Anyway, all the info was great. Thanks!

Regards,
tltan