Determine antibody concentration - (Feb/26/2009 )
If you desire to work in little volumes you should use a NanoPhotometer from Implen, it is flawless for protein trials since their expertise is not counting on exterior stress of your samples. BCA, Lowry, etc. is furthermore likely since the equipment works with corvettes, aslo..
You might also take into account that the IgG bands might not always so up at there usual spots on the gel. As some antibody can be aggregated, fragmented and so on
mdfenko on Wed Mar 4 16:35:49 2009 said:
I have a question regarding IgG. When running the abs on a protein gel, the heavy chain gives a strong band while the light chain only gives a weak band. Is this normal?
Second, I have used the protein chips from Agilent to use the Bioanalyzer (Agilent) to get an idea of the purity as well as concentration. Also here, the heavy chains constitutes approx 80% of the total amount of IgG while the light chains only approx 16%. I'm just getting confused here, shouldn't there be a 1:1 ratio or am I missing something obvious?
Using the bioanalyzer the heavy chain and the light chains gives separate concentrations (since they are separated and measured individually), for example in one sample 130ug/ml heavy chains and 25ug/ml light chains. Should I then combine the two concentrations, and then get the total IgG concentration?
the molar ratio may be 1:1 but the actual amount of protein will be different because they are not the same size.
what you found sounds okay and, yes, you can add the totals (not the concentrations) together.