Correcting phospho-protein with total protein - (May/01/2007 )
Hi,
If im using an phosphorylated antibody, such as phospho-MAPK (mouse) and want to correct the signal for total amount of protein in each extract, unphosphorylated antibody MAPK (rabbit); how do i do it ? What i did now is that after the first blot, i put secondary antibody (anti mouse) and detected bands. But now, to correct for total amount of protein, do i just put the unphosphorylated antibody, then anti-rabbit for second antibody, and detect bands again ? If i do this, how can i be sure that the second scan only detects total protein and not the phosphorylated one, since they have the same molecular weight ?
Is stripping required ?
If not, does rinsing with TTBS between the phosphorylated antibody and unphosphorylated antibody take some of the first signal off ?
How does it work exactly.
Any help appreciated.
Thx
The antibody for the total protein will detect the phosphorylated AND the non-phosphorylated form of the protein. However, the phospho-specific antibody will recognise only the phosphorylated form.
So, if you want to blot the exact same extract, you must strip your membrane before reblotting it with another antibody. I would recommend doing another gel though, since stripping is not always efficient.
As for the other questions, washes with TBST does not interfere with antibody-protein complexes, so rinsing with TBST should not take the first signal off.
If im using an phosphorylated antibody, such as phospho-MAPK (mouse) and want to correct the signal for total amount of protein in each extract, unphosphorylated antibody MAPK (rabbit); how do i do it ? What i did now is that after the first blot, i put secondary antibody (anti mouse) and detected bands. But now, to correct for total amount of protein, do i just put the unphosphorylated antibody, then anti-rabbit for second antibody, and detect bands again ? If i do this, how can i be sure that the second scan only detects total protein and not the phosphorylated one, since they have the same molecular weight ?
Is stripping required ?
If not, does rinsing with TTBS between the phosphorylated antibody and unphosphorylated antibody take some of the first signal off ?
How does it work exactly.
Any help appreciated.
Thx
you should strip to apply a second Ab for the same polypeptide; better use first the Ab with the estimated lower signal to avoid intereference of non-stripped rests of the other Ab