serum samples for western - (Sep/15/2006 )

hi
I have to perform a WB using human serum samples, but I don´t know if this needs any special treatment. I did a bradford and decide to dilute them 1:10 before adding laemmli buffer. Should I remove albumin from serum? Does somebody know if there is a specific protocol for this sample? I can´t find anything anywhere

I never performed WB on serum, however I don't think there is a special treatement?

the only thing that could ennoy you, would be if you use a human primary antibody (because you would have some human Ig in your sample that would be recognized by the anti-human Ig)

Let see what others say.

since the major proteins in serum are albumin and gamma-globulins, most other proteins are in low abundance relative to albumin and globulins. when you perform the protein determination that is what you are reading. by diluting you are making it more difficult to find your protein of interest.

you only need to remove the albumin if your protein is too close to and gets overshadowed by it.

I think it´s not clear what´s the purpose of separating serum; as mdfenko says albumin may disturb detection of similar sized polypeptides by its high abundance and causes shift effects what makes determination of apparent Mw difficult.

Bradford: Although BSA is not really to recommend for calibration, in this case it is fine.

thank you mdfenko.

I´ve read that for WB it´s recommended to load about 0,5mg/mL - 1mg/mL. Is this correct? what happen if you load more than this?

If I don´t dilute the serum, I will be loading too much protein (specially too much albumin and Igs).

If I remove albumin I would be enriching my serum in low abundance proteins, isn´t it? when you say "too close" you wanna mean if my protein and albumin have a similar weight?

thanks and sorry for so much questions

PD: excuse my english

the amount of protein you load is determined by the binding capacity of the membrane and the complexity of the protein mixture. the more different proteins in the solution, the more you can load (as long as they separate on the gel).

if you don't dilute then you will probably overload the major proteins but not the discrete minor proteins. the excess major proteins, per unit area of the membrane, just won't bind.

if you remove albumin then you will probably also remove some of the other proteins, as well. it is very difficult (if not impossible) to selectively remove albumin without sacrificing some or all of the lower abundance proteins (i know, we've tried).

yes, i meant too close in migration in the gel (molecular weight in sds, pI in ief).

it's not too many questions, it's important to understand. and your english is fine (mine may be awful).

thanks guys!
I´ve been investigating, and people next door use some kind of spin columns with antibodies against albumin (from Beckman Coulter , I think) to remove albumin from serum. I can try that columns and compare my WB with and without albumin and see how that affects to my protein.
My membrane is made of PVDF, I think this has a good capacity of binding proteins, hasn´t it?

thanks again

PVDF is fine, and commonly used; good luck!