I need to compare the concentration of a protein in serum and plasma samples to matching CSF samples. I preferably don't want to purify the samples but I am concerned about other proteins. I have specific antibodies that work very well in other immunoassays. Any help is appreciated
The whole point of SDS-PAGE (the first stage of western blotting) is to separate proteins. If you are really worried about specificity, you could run a 2D-electrophoresis and then blot. If a protein has the same size and pI as another, and also binds to the same antibody, it most likely is the same protein.
Btw, what does "other assays" mean? Antibodies that work well with native protein, say, in immunofluorescence or immunoprecipitation may not bint to the denatured proteins on a blot. The problem is especially acute with monoclonals.
Running serum in SDS-PAGE presents no special obstacles except the high concentration of albumin, which can obscure the presence of proteins present only in small concentrations. Should that present a problem then there are special albumine-binding columns commercially available, but I'd use them only if absolutely necessary.
Hi Catarina,
It should be fine for you to run your samples without purifying them first (although western blot can sometimes be a bit of a finicky assay). However, if you want to do anything quantitative with your western blots, it is important that your normalize your protein content between samples before performing this test.
I recommend for you to perform some kind of protein quantification assay, such as a Bradford Assay or the Pierce BCA protein assay. Once you know how much protein you have in each sample, you can create working stocks of your samples that are all at equal protein concentrations to one another. You can then run a Western blot on these normalized samples.
I hope this helps.
Regards,
Lindsay
As per the other comments,
Yes is fine!
However I suggest an optimization phase (loading volumes, type of loading buffer, running and transfer conditions as well as ab concentration) in order to have reliable and reproducible results.
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