I am comparing the protein expression levels in subcellular fractionations (Cytoplasm and nuclear). If I load an equal volume of both extracts (15 ul each to individual wells), is this still valid to compare? I am using the loading control GAPDH for normalization. Or should equal amounts of protein be loaded for quantification of the blots?
Thanks in advance!
Hi Gowthami Mahendran
Generally, it is recommended to load the same amount (ug ....) of each sample not the same volume. So, you have to run a protein quantification assay before performing WB.
Best...
So this is a bit tricky. Normally you would load equal amounts of protein if you are doing a comparison, but in your situation I think not. Since you are comparing two cellular compartments I think you are are wanting to quantify how much of your protein is in each compartment. In that case you would need to load equal volumes of the each cellular compartment. But whether this is equal volumes or not depends upon how the fractionation was done. If after fractionation you have equal volumes of nuclear and cytoplasmic fraction, then equal volumes would be correct. However if you are asking the question what percent of the total protein in each fraction is your protein of interest, then you would load equal amounts of protein. But you need to think through the precise question you are trying to ask.
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