For 30 µg protein loading in each lane, concentration of one protein is 1.97 µg/µl and another is 1.41 µg/µl, that means for first one would be 15.2 µl and 2nd one is 21.2 µl.
How to load equal volume with different concentrations in each lane?
Pre-dillute higher conc sample with 1xSDS sample buffer to match lowest conc sample
Is it possible to use RIPA lysis buffer instead of 1X SDS buffer @Sven Hennig
yeah sure, pre-dillute to a common low concentration and prepare your samples all equally.
You might still get little deviations, but therefore also test for GPADH or b-Actin as house keeping genes as loading controls in your western to make sure their intensities are even.
Use gas-tight injection syringe (Hamilton) . Also, sonicate your lysate before loading. It reduces viscosity by fragmenting genomic DNA.
I would say precipitation is not required. You can take the exact volumes of each protein, add the same amount of a 2x Laemmli buffer, and then in a second step a 1x Laemmli buffer up to the volume you want to apply to the gel. @Osamu: It is always good to use a HAmilton syringe. Before loading I would do a short spin of the boiled samples to spin down unwanted particles clogging the gel lanes.
You may want to dilute your concentrated samples with 1x running buffer or water to equalize their volumes prior to loading.
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