cracked gels, no thanks, please help!!
Bassuk at U.Washington.Edu
Sun Jan 15 20:51:28 EST 1995
In article <01HLSNOOLPUQ00AB6D at msvax.mssm.edu>, LAURETTE at MSVAX.MSSM.EDU (STRUCTURAL NEUROBIOLOGY LAB) says:
>Dear Protein/Peptide People-
> I am trying to complete some preliminary
>data for a grant due in one week.
> My problem is I am doing a tryptic digest and attempting
>to run either the Schlagger and von Jagow Tris/tricine procedure
>OR the laemmli modification of Okajima with 17-18% gels.
> Samples appear fine by counts BUT the gels CRACK
> My question is: how to prevent gels from cracking
>[i am using 35S-Met labeled peptides [2-5.5 kD] upon
>drying. if i soak in glycerol [what %] won't i get diffusion?
>i am looking for any and all comments and suggestions.
>frustrated young post-doc
Your point about diffusion is correct. Therefore, I suggest you fix your
polypeptides in the gel first. 45% methanol or ethanol should be OK.
10% trichloroacetic acid is even better, unless your peptides are soluble
at this concentration. Therefore, I would fix in 45% methanol/10% acetic
acid (30 min for 1 mm thick), and soak in 10% TCA (30 min for 1 mm),
5 x water (5 min each), and then once your water rinse is no longer
strongly acidic (< pH 5), then soak in 17-18% glycerol for 2 hours.
Your gels won't crack (keep drying temp < 60 and use a good vacuum
<100 mTorr). And, you will have done your best to minimize diffusion.
Besides, it is better to have the figure for your grant then not to have any
data worth photographing or scanning.
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