1
$\begingroup$

I'm currently trying to understand the concepts of SDS-PAGE. Every protocol I've read says to use 5X for sample buffer and 1X for running buffer, but I don't really understand for what reason? What are the negative consequences if I don't?

$\endgroup$
  • $\begingroup$ Please give the composition of your buffers. You can't expect us to know what they are and if we don't we can't give an answer to your question that is of use to anybody. $\endgroup$ – David May 11 '17 at 21:42
3
$\begingroup$

The reason why the sample buffer is more concentrated (typically 2x or 5x depending on your protein concentration) is its dilution when you mix it with the sample. You mix 4 parts of your sample and 1 part of 5x sample buffer, so that the final concentration of this buffer is 1x.

If you use 1x sample buffer together with the same amount of sample, the final concentration of the sample buffer would be 0.5x, if you do the same 4+1, it would only be 0.2x in the end.

This dilution is too strong and the buffer will not fulfill its purpose, the complete denaturation of all proteins in the sample, so that their distribution on the gel depends only on their molecular weight and not also on their tertiary/quarternary structure.

Using a 5x buffer allows you to add a lot of sample when diluting it to 1x without changing the concentration of the protein too much. It also allows to load larger amounts of sample compared to 2x buffer, interesting when the protein concentration is low.

The running buffer is not further diluted by adding sample, so that it has to be diluted to 1x in order to have the right ionic strength. Since these are often used in big quantities in the lab, it is more convenient to prepare concentrated buffers in smaller quantities and dilute them before usage than preparing big tanks of buffer.

$\endgroup$
  • $\begingroup$ Worth adding that the reason for using e.g. 5x sample buffer is so that the sample can be brought to 1x without greatly increasing sample volume and thus decreasing protein concentration. In many situations you may want to load as much of your sample protein as possible in the well. $\endgroup$ – Alan Boyd May 17 '17 at 18:59
  • $\begingroup$ @AlanBoyd Good point. I do this often but don't think too much about it anymore :-) $\endgroup$ – Chris May 17 '17 at 19:00

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.