Both columns are pre-treated with BSA, in one column there is going on some precipitation or beads have some particulate material. or due to pH effect or due to some contamination your column is not working. Particulate artifacts usually chock the column if remain unused for longer time or if it is kept in low temperature elution effect may be there due to over saltation. If it is preparative column then take out beads, and a fresh them then re-pack.

what do you mean by pre-treated with BSA?

I must say I lost lots of activity with HiTrap desalting column with one my enzyme as well. In fact, I lost activity also on another HiTrap column (I think it was the Blue Gel). But I haven't tried another desalting column for the purpose. And the enzyme was very unstable, so I thought it was rather because of the enzyme, than because of the column.

To answer both of you. The columns are disposable so the media is fresh every time it is used. I would not believe there is precipitation going on in one column and not the other but, possibly, if the higher pressure might play a part...regarding temperature variation I have tried columns store in refrigerator and in room temperature with similarly bad results so I don't expect this to be the case.

Pre-treated with BSA means that a volume of BSA-PBS solution was let to pass the column before buffer exchange to fill any unspecific sites and decrease protein retention in the column. This is something we do as routine to increase output through these type of gel-filtration columns (and it usually helps a lot).

Hi Emma,

Can you tell us what salt concentration/buffer you are using for the buffer exchange? 'Desalting' can also be a bit of a misnomer as many people will use it for buffer exchange into different buffers that may still contain similar salt concentrations. It is rarely used in the complete absence of salts.

If your buffer does not have sufficient ionic strength your labelled proteins will stick through electrostatic interactions regardless of whether there is BSA present or not, In fact if you have sufficiently high salt concentration you should not need to add BSA to prevent protein retention on the column.

And is this step to remove the isotope label? There should be a number of 'clear' fractions between your protein peak and second salt/free label peak that should contain no protein or radioactivity (or very little). If there are not then you either have too large/too concentrated sample or insufficient salt to overcome the electrostatic interactions.

Hi Nick and thanks for the answer.

I use PBS (Phosphate buffered saline) from tablets so I think the ionic strength should be sufficient, but maybe it isn't (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4)? This I can try to change and see if it makes a difference. The idea of the desalting step is to purify my labeled compound from un-attached nuclide, but even if I start with an already purified version (from gravity, NAP5, since this works well), a lot of my activity (>25%) stick in the HiTrap column. My sample concetration is not very high (ca 0.6 mg/ml) and volumes are not large (0.7-0.8ml).

Emma, what is the size of the protein you're working with and what kind of labelling do you perform?

An alternative to desalting (size exclusion chromatography) might be precipitation with ammonium sulphate (AS): Add solid AS close to saturation, spin and wash with saturated AS, until no radioactivity is washed out. Then drain the pellet well and dissolve it in your favourite buffer. Some added BSA or similar as carrier protein might increase recovery (it also will in gel filtration).

HTH

Hi Emma,

We have used this resin for many buffer exchanges with many different proteins and it is hard to predict which ones will cause problems.

If you add your unlabelled protein to 1X PBS solution do you see any signs of precipitation? If not try it also with 2xPBS, we have previously used 2XPBS for a problem protein during buffer exchange  to prevent protein retention, then you only have to dilute the PBS and you don't have to worry about additional buffer components.

If you do get precipitation it could be that your protein just doesn't like phosphate so you may have to switch to another labelling buffer -just make sure the buffer component doesn't contain anything that will react with your labelling reagents, e.g.  tris has an amine group.

if you still have problems in higher ionic strength buffers you may also consider using a detergent in your buffer if it will not interfere with your assays.

Thank you Nick! I dont see any precipitations in PBS so I'll definately try to use 2xPBS. If this simple solution solves my problem I will be extatic!

and thank you Wolfgang! It is an antibody and I am glad to hear about this alternative method that you present although I have to do it online so in this particular case it isn't an option. But if I get trouble in manual preparation some other time I will definately keep it in mind. 

Thank all of you for answering and have a nice weekend! :)

Emma,

Good question, 

And  on behalf of the TATgroup Many Thanks to all of you engaging and giving valuable input to the problem.