I am measuring protein conc by Bradford assay, r2 value is >98, conc of protein prepared is 2ug/ul.
Does your concentration value fall within the calibration curve?
Do you load the same amount for each lane?
Your question is not clear.
, Dear Riccio, yes it does, the orblem is we eavluate the protein conc by usual BSA Bradford assay, after that we prepare protein sample with SDS to a final conc of 2 ug/ul, but unfortunately, i got the problem of unequal internal control, so none of my expt is valid, hope now you can understand..
Estimate the total protein.Calculate the total protein to 30ug. Let us say, if you get a protein concentration of 2ug/ul; take 15 ul of total protein and add 7.5 ul of 2X SDS dye. Boil for 5 min at 95 degrees. Brief spin the samples and load them carefully. Do not spill the samples.
Note: Loading lower volumes will result in improper loading. Make sure, you make up the volume to around 20-25 microlits.
Unless you are loading different cell lines, you should not be getting a huge variation in your loading control. If the different samples are clonal populations of the same cell line also, the same problem will come up. You can quantify the density of the actin or tubulin band in each sample. Normalize it to a certain value or volume. And load the volume of each sample that would give you equal amounts of actin or tubulin across the board.
However this is an imperfect solution, and you should use this only if there is no other way around.
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