Glycoproteins and IEF gels

[Halleys_Come]

Hi everyone,

 

I have a large glycoprotein (runs at 140KDa on a reduced denatured gel) about have of its apparent molecular weight is sugars. I produce this protein in Chinese Hamster Ovary cells K1 which should add Sialic acids to the protein. Hence, making the pI of the protein slightly acidic probably 4-5. However, I have run the gel 3 different times with the same protein and got three different answers.

The first time the protein was in 0.3M NaCl, 50mM Citric Acid pH5 and the pI came out between 4-5 great I thought, I will be able to repeat this easily for my thesis.

 

However, the second time I had changed the elution buffer on the size exclusion column to 1M NaCl, 50mM Hepes pH6.5 and this time the pI came out around 6-7.5. So this put me in a quandry is it following the buffering capacity of the buffers or is it, as some people have reported, being made more alkaline due to an increase in salt.

 

So I decided to dialyse my protein into a 10x diltued IEF loading buffer (pH10.3) and then run the sample, therefore the salt or the buffer can't affect the result - however now it runs with a pI of 7-8.5. I don't know if this is correct or not.

 

All the theoretical pI databases give the protein sequence (without sugars) a pI of around 7.75ish

 

Can anyone help, this one of my last experiments, I want to finish this thesis soon,

 

Any help would be gratefully recieved,

 

Thanks