Home >
Community >
How to analyze phosphorylation shift by western blot?
Upvote
30
Downvote
+ Biochemistry
+ Proteins
Posted by
Mustang Mark
How to analyze phosphorylation shift by western blot?
The run length of bands in an SDS-PAGE (which your western is probably based on) depends - at least in theory - only on the size of the protein: SDS binds to proteins with a fixed size ratio and thereby provides a negative charge based only on size. Unless you have reason to believe that your protein is an exception from the usual (which might be indicated by things like not running at the expected size) you will very likely not be able to see a band shift (change in run length) based on presence or absence of phosphorylation in a standard western blot.
Things you could try to detect the phosphorylation:
Use phospho-specific antibodies. These are often expensive, tricky to use or just not available, but this is the only ting I can think of working with a standard western blot setup
run a native-PAGE, that way your run length depends on shape, size & charge of the protein. I still wouldn't expect a single phosphorylation to be cause a visible shift for a 150kDa protein, but you can try. This might require using longer run-time of the gel to get really high resolution for the size you are looking for
The run length of bands in an SDS-PAGE (which your western is probably based on) depends - at least in theory - only on the size of the protein: SDS binds to proteins with a fixed size ratio and thereby provides a negative charge based only on size. Unless you have reason to believe that your protein is an exception from the usual (which might be indicated by things like not running at the expected size) you will very likely not be able to see a band shift (change in run length) based on presence or absence of phosphorylation in a standard western blot.
Things you could try to detect the phosphorylation:
Use phospho-specific antibodies. These are often expensive, tricky to use or just not available, but this is the only ting I can think of working with a standard western blot setup
run a native-PAGE, that way your run length depends on shape, size & charge of the protein. I still wouldn't expect a single phosphorylation to be cause a visible shift for a 150kDa protein, but you can try. This might require using longer run-time of the gel to get really high resolution for the size you are looking for
@tyersome I guess it is possible in some cases even though it normally shouldn't happen - I edited the answer. Also note that the authors of that paper were "using a modified acrylamide/bisacrylamide ratio" for the gels to get a visible separation of the two bands.More
I agree that observing a shift for a 150 kDa protein may be difficult, but shifts due to phosphorylation can be seen for some proteins on SDS-PAGE gels. See for example More
The run length of bands in an SDS-PAGE (which your western is probably based on) depends - at least in theory - only on the size of the protein: SDS binds to proteins with a fixed size ratio and thereby provides a negative charge based only on size. Unless you have reason to believe that your protein is an exception from the usual (which might be indicated by things like not running at the expected size) you will very likely not be able to see a band shift (change in run length) based on presence or absence of phosphorylation in a standard western blot.
Things you could try to detect the phosphorylation:
The run length of bands in an SDS-PAGE (which your western is probably based on) depends - at least in theory - only on the size of the protein: SDS binds to proteins with a fixed size ratio and thereby provides a negative charge based only on size. Unless you have reason to believe that your protein is an exception from the usual (which might be indicated by things like not running at the expected size) you will very likely not be able to see a band shift (change in run length) based on presence or absence of phosphorylation in a standard western blot.
Things you could try to detect the phosphorylation:
More
VOTE
VOTE
VOTE
VOTE
VOTE