Try with IPG strips of pH 5-8 and 3-10.

Dear Masood:

I have used a well established protocol for yeast sample prep. (with 7M Urea and 2M Thiourea in the IPG Rehydration buffer), and the recommended focussing protocol for 7 cm drystrips (3 phases). Further I have tried out both over-night rehydration of the strips with the sample and sample cup loading, but results were similiar. It is only this one protein that shows a smear; other proteins are nicely looking..

Dear Bimal:

I have already tried 3 other stripes, but the smear for this one protein remains..

Thank you

Dear Daniela,

What is the expected pI of your protein of interest? Maybe your pH range is not proper. In overall, your blot looks nice, the spots between pH5-6 are nice. In general, it is difficult to deal with extreme pI proteins. You can always clean up your sample to get rid off all the agents that could spoil your running pattern, such as salts, nucleic acids or lipids. Try to clean up your sample with one of the precipitation methods, but first it is better to calculate the expected pI of your target protein.

Istvan

Dear Istvan

Thanks for your answer. The expected pI is 5.8 and I know, that I can see isoforms in the acidic region because of the phosphorylation status, so the range of pH 4-7 should be fine. I will try to "clean" the sample a bit more and see what will happen to the smear..

I see, so if you detect that acidic spot (around pH 4, phosphorylated protein) only upon a treatment which induce the phosphorylation of your target protein, I think the quality of your blot is good enough. You can check many 2DE papers and you will see that both the acidic and basic ends are always a bit smeary, especially if you have a lot of protein there. Alternatively, try to use phospho-specific antibody, if it exists. Anyway, good luck!