I added 4x leammli buffer to my protein samples and DTT. After heating it up to 95°C for several minutes I placed in in the fridge. I planed to run the samples the next day. When I heated them up again for 5 min next day I meantioned afterwards that there were precipitates within the sample. I loaded it on the SDS-gel anyway.The gels were awful because there were Protein left in the slots and the fronts were not straight. Does somebody know how to resolve the protein in the samples?
How pure was your protein extract? Usually when there is lot of DNA in the sample (like when you directly put cells and laemmli buffer) you can get real trouble loading (very gummy and viscous) and during migration the well will be deformed and the profil will be rubbish
It should have been quite pure. I extracted them using RIPA buffer and the lysate was clear.
The SDS in your sample buffer may crash out of solution in the fridge. That could be you precipitate. Did you mix you samples well before loading them? That may help.
You should always freeze your samples if you are not planning on running them right away. Also, the salt concentration may have been too high - did you make your final solution 1x Laemmli? If so, I would suggest trying to use a final concentration of at least 2x Laemmli.
Hope this helps!
As mentioned you can have this kind of problem if your samples contain high amount of DNA. Usually you can resolve it by adding and endonuclease (OmniCleave endonuclease is very good for that).
As Gaël Nicolas said, the DNA is the problem here. RIPA usually results in DNA with nucleosomes (Histones). When you heat it up it denatures...when it denatures it expands...but still hold togather by DNA strands. As Gaël Nicolas suggested, endonuclease is a good but expensive option. You can sonicate the samples before you clarify the lysates at high speed spin. Then use the supernatent for SDS-PAGE.
>As Gaël Nicolas suggested, endonuclease is a good but expensive option.
Yes ... very expensive!
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