phosphorylation of a protein - (Nov/29/2006 )
my protein is 62KDa. it has several phosphorylation sites. so phosphorylation should increase its mass by 80 Da right? there are papers that say that 64 and 66 KDa forms are the phosphorylated forms of this protein. so it must mean that there are around 25 sites that are phosphorylated 
. but only 3-4 sites are known and spoken of. 
please can you explain this to me? i am sure i am mixing up some things as usual. 
I don't have an answer for you but I was wondering where you found the number (80 Da) for the mass increase due to phosphorylation.
. but only 3-4 sites are known and spoken of. please can you explain this to me? i am sure i am mixing up some things as usual. I still have the same problem (already discussed in here!). But I´m still wondering about a shift caused by a few phosphorylations.
A little fancy play of thought:
Protein: 62 kDa
One Phosphorylation +98 Da
SDS: 288 Da
SDS/protein: 1,4g/1g
One protein of 62kDa will bound 86800 Da SDS, this means to one protein we´ll adding 300 negative charges. Compared to one additional negativ charge of one phosporylation this will never change the behavior of the phosphorylated species in the SDS-PAGE.
Still confused...
my protein is 62KDa. it has several phosphorylation sites. so phosphorylation should increase its mass by 80 Da right? there are papers that say that 64 and 66 KDa forms are the phosphorylated forms of this protein. so it must mean that there are around 25 sites that are phosphorylated
. but only 3-4 sites are known and spoken of. please can you explain this to me? i am sure i am mixing up some things as usual. I still have the same problem (already discussed in here!). But I´m still wondering about a shift caused by a few phosphorylations.
A little fancy play of thought:
Protein: 62 kDa
One Phosphorylation +98 Da
SDS: 288 Da
SDS/protein: 1,4g/1g
One protein of 62kDa will bound 86800 Da SDS, this means to one protein we´ll adding 300 negative charges. Compared to one additional negativ charge of one phosporylation this will never change the behavior of the phosphorylated species in the SDS-PAGE.
Still confused...
are you definitely sure that there is only one mol phosphoryl per mol protein?
I agree that more the mass than the negative charge of phosphoryls make the shift in 1D; what about 2D? here you should better monitor phosphoryl-dependent shifting ( for pI) as pI depends on degree of phosphorylation; there are online-tools to caculate the pI on the bases of primary sequence and number of introduced phosphoryls...
post translational modification masses
yes i have the results of 2D too. spots do migrate towards the acidic part of the gel. they are more acidic than the native protein. so, what confuses me is their mass. on 2D and on western they are around 66 or even 70 kDa
. that simply isnt possible, there are no such sites in the protein. but what is more confusing to me is other papers already published results that 66 kDa protein is 62 kDa protein with 3 phophoryalted sites. how on earth they have calculated that??? i can;t understand it. Sorry for the late post, but I read here that phosphorylation can interfere with SDS binding and thus reduce the negative charge of the protein in SDS PAGE, leading to a disproportionate decrease in mobility and thus increase in apparent MW. But the effect is variable.
http://www.bio.net/bionet/mm/methods/1995-...ber/036038.html