I've been trying to obtain a 2D-PAGE from a protein extract, but I haven't obtained an adequate result. So far, I've obtained gels with poor proteic content or with an artefact in the lower molecular weights (like a perfect line). What could be the step I'm failing at?
Can you elaborate: used conditions, gel density, sample type, used loading buffer..
Mateo Glumac
I used a pH 7 -10 IPG strip with IEF program of 250 V - 40 min, 4000 V - 3 h and 4000 V until 16k V/hour
It is a 12% polyacrylamide gel
My sample is a an engorged tick total protein extract.
I didn't used a loading buffer, I just deposited my IPG strip directly into the separative gel and then cover it with .8% agarose.
Program sounds fine, so is the gel thickness. How do you solubize your protein? Is it just a lysate in the last lysis buffer or do you use buffer solutions fro some commercial kit?
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