dear all
my problem is that, when i mix my samle buffer to protein (that i want to migrate), the muxture became coagulated, some forum said that because of SDS, but i dont know how to evoid this problem, does this mean preparing a samle buffer without SDS ??! or there are other solutions??
what i observed that when i heat the coagulated mixture it beacame liquide but i have to load it fastly, even with this methode my sample coagulate in the tips and i can't load only a tiny quantity and subsquently no band or a very weak intensity and deformed band.
I don't know if some one crossed this problem, thank you in advance for your help
greatfully.
leila
there could be two reasons for it:
1: you have highly concentrated protein sample, if yes, dilute it to get good bands. dilution will solve your problem of having viscous samples.
2: your protein extraction protocol is not complete and you are having cell organnells in it, specifically nucleus and cell debris. if this is the reason you will have highly viscous protein sample. so take a aliquat of it and centrifuge it at 1000g. take supernatant and this should solve your problem.
Hi!
I had a similar problem and I discovered that it is the DNA the one forming the clumps... so after I boil the samples I cool them in ice or I add DNAse to my lysis buffer. Although teh first problems came with concentrated bacteria samples, I had the same problems with lymphocytes... and it was always the DNA...
About centrifuge, well, I would not do it... I saw that because of the stickiness of the DNA, some of my samples were not reproducible when I centrifuge.. I lost a lot of the proteins...
I hope this helps you...
I would suggest to use benzonase instead of DNase, in case you are loosing lots of sample. centrifugation is a standard procedure. benzonase is more efficient than DNase and and degrades all forms of DNA and RNA.
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