how to prepare blood sample for western blotting? - (Nov/18/2007 )
do i need any lysing step if i want to blot protein (growth factors) in blood sample.
how do i prepare my blood sample for western blotting?
This is what I have done several times to study a secreted protein in serum (I suppose it´s the same for a growth factor because they are normally secreted).
1.Centrifugue blood (3000 rpm 15 min) to obtain serum (cells will be in the red pellet) where you have your protein of interest.
2.Take the serum and discard the pellet.
3.Perform an assay to determine the protein concentration in serum. I did Biuret better than Bradford because the prot concentration in serum is high. For human serum I obtained 77 mg/mL.
4.Dilute your sample in Laemmli buffer to the amount you want to load in the gel.
5.Do the WB as usually.
6.Note that the elevated concentration of albumin in serum could mask your protein. I recommend that, to obtain reliable results, ensure that the molecular weight of your protein is not very close to the MW of albumin.
how much is very close?
how much is very close?
In my case, with a minigel, my protein weight 30 KDa (albumin is 66 KDa I think) so I didn´t have problems, they were separated. I suppose if your protein is around 60-70 KDa, it could be masked. I think it depends on the size of your gel and how it resolves proteins. But I don´t have too much experience about protein electrophoresis, perhaps other members could advice you better.