Why sometimes the IPG strip at some portion shows a swollen part after first dimension run which results in bad resolution of proteins in the SDS-PAGE gel after the second dimension run?
The IPG strips may sometimes show swollen portions after the IEF run, which is due to high salt content/high ionic strength of the samples loaded. This can be avoided by protein precipitation methods such as TCA precipitation or others followed by proper washes which will remove salt and other non protein components of the sample.
The IPG strips may sometimes show swollen portions after the IEF run, which is due to high salt content/high ionic strength of the samples loaded. This can be avoided by protein precipitation methods such as TCA precipitation or others followed by proper washes which will remove salt and other non protein components of the sample.
Hi Dear Bimal
The comment by Anjana is completelty true. However, I can also recommend using of TCA/acetone or only acetone precipitation to remove high salt concentration.
If possible, it is better to use salt free buffer for homogenization. For example, Tris is better than PBS.
Finally, although, the comment of Pavel is approximately true, but I don't agree with his completely. It is better to use wicks which soaked in the SDS solution to prevent induction of disulfide bands.
The best method that I used and found reliable results are presented in the following my article:
Electrophoresis 2014, 35, 3331–3338.
I also agree that high salt concentration can be responsible for this effect. They used to precipitate at ends of IPG-strips. TCA/acetone- or just acetone-precipitation can help you as well as changing the paper wicks.
I agree with Anjana devi Tangutur that high salt concentration can cause this. However, even after acetone precipitation this happens.
As Pavel Májek mentioned. I sometime interrupt the run and exchange the paper wicks with fresh ones. However the problem is not solved by doing that. I would like to add that many times some researchers do not add the paper wick at all and go for IEF. I could know from one of my scientist colleagues that while she was working in Germany, in their lab paper wick is not used at all, which means paper wick is not essential. However, such labs must be maintaining a very very high level of QC.
I found the same problem when I homegenized the samples directly in the rehydration buffer itself; as rightly mentioned by Masood and Karolina that PBS and other salts could be responsible.
Still searching for a solution.
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