4 replies to this topic

[Inmost sun]

We had the problem that a negatively charged (low pK) protein did not run in SDS-PAGE.  I guessed that it would make no difference if boling in Laemmli buffer or not before loading onto gel. But what we experimentally found is if sample was boiled in Laemmli buffer it didn´t run. Instead it run without Laemmli buffer.

Who has an explanation for this?

[bob1]

That's pretty odd as the Laemmli buffer is supposed to wrap the protein in negative charges (from the SDS), which allow it to migrate in the electrophoresis - however, I suppose you could look at it in physical terms - the negatively charged protein is repelling the negatively charged SDS  so there is no benefit from boiling in Laemmli.

[Inmost sun]

thanks for answer but why blocks Laemmli buffer the running of the negatively charged protein?

[bob1]

Perhaps there is something in the Laemmli that is + charged - so it binds to the negative charges on the protein, thereby neutralising the charge and preventing the protein from migrating?  Just a possibility, I don't know for sure.

[mdfenko]

it may be that the boiling is causing the protein to aggregate.

try running the samples with sample buffer without boiling.

you can also try heating the sample at ~65C for 10-20 minutes instead of boiling.