Selected from Jim Woodgett
A question to somebody with experience: How much does the
; phosphorylation of a protein affect its movement on SDS PAGE? Is
; the difference significant, say could it be that the protein seems
; about 2000 da larger than it is supposed to because it has
; been phosphorylated? Yes and no. It all depends on the particular protein and the gel system you use.
The mobility shift is an artefact caused by a reduction in SDS-binding to the protein (presumably close to the
phosphorylation sites). However, in some cases a single phosphorylation site can cause a 10 kDa shift, in others
multiple sites have no effect. SOme gel systems allow high resolution, others lose the differences.
You can't rely on a shift being due to phosphorylation (other posttranslational modifications can shift mobilities)
and you can't rule out phosphorylation if a shift doesn't occur. If you suspect phosphorylation try
the following experiments:
1. Treat your protein sample with purified phosphatase (alkaline or acid or PP-1 or 2A) +/- pyrophosphate
to inhibit the p'tase. If the band shifts to a faster mobility you're likely dealing with phosphorylation.
2. 32P Phosphate label your cells and immunopurify the protein.
3. If its cloned, express the protein in bacteria. Often proteins aren't modified in bugs
which thus provides a source of "apo-protein".
4. If you have it clean enough and its not too big try FAB-MS as this generates a more reliable mass.
5. If you suspect the protein is phosphorylated on tyrosine immunoblot it
with anti-pTyr antibodies (commercially available). Examples of shifting proteins:
c-Jun phosphorylated at serine 63/73 -> 39 kDa to 43 kDa Erk-2 phosphorylated at tyrosine and threonine
; 42 kDa -> 44kDa Examples of non-shifting proteins:
c-Jun phosphorylated at three C-terminal sites c-Myc phosphorylated at numerous sites (very small change)
Check out Methods in enzymology vols 200 and 201 for techniques in protein phosphorylation.
There's also a new Practical Approach book on the same topic.
Jim Woodgett
