Protein precipitation on column

[nashnash]

Hi

I'm attempting to purify a protein by ion-exchange, the protein binds to Q-sepharose only in 2mM (phosphate) buffer. But the unbound proteins precipitate on the column during washing; this causes my protein of interest to elute with significant loss in resolution. I ve found that including 1mM DTT solves the problem to some extent, but it becomes too expensive to add DTT in all the buffers. Could anyone suggest other additives or new ways that could help counter this problem.. or is DTT a must?

And what is the best way to clean the column to remove the precipitated proteins? (Will 2 bed volumes of 2M NaCl + 2 vol 0.5N NaOH + lots of water suffice?)

[DaveM]

Hello,

I would say that 2mM phosphate is very close to not being a buffer at all. What pH are you using? Did you try changing the pH to try and raise the buffer strength, and have better binding? If your buffer conditions are not adequate then your proteins will be binding to your column by different mechanisms.

To your questions, can you simply add the DTT to the sample and incubate for some time (30mins?) before applying to the column? I'd say DTT is one of the cheapest additives for generating a reducing environment. TCEP (tris carboxy ethyl phosphine) is another. Other additives might help, but it does depend on why the DTT helps. For instance, if it is reducing a disulphide bond to give a free cysteine, then you may do well to protect this cysteine or mutate it out (if this is practical or desirable).

For cleaning the column, I'd always do salt, base and acid washes, with water in between, and multiple switches between base and acid. If you set it up to run overnight, it shouldn't hold you up too much.

DM

[nashnash]

Hi

I need to clarify certain things here. I use 2mM phosphate buffer at pH 6.5; primarily because the protein doesn't bind to Q-sepharose with 10mM buffer (till pH 8!) But it binds with 2mM buffer, and at pH 6.5 I'm able to get my protein purified upto around 85% (i got 3 bands, 1 prominent and the other 2 faint, on PAGE after silver staining).

When I increase the pH, other proteins elute too, and resolution is not very good. So I prefer to stick to the same conditions as mentioned above. What I would like to know is

1) How can I improve stability of my protein in 2mM buffer, apart from adding 1mM DTT?
2)Can I load my sample in 2mM buffer and develop the column with 10mM buffer? (especially because addition of 0.1M NaCl to 2mM buffer decreases the pH by 0.5 units!)
3) Any other modifications/suggestions (reg flow rate etc.)?

[DaveM]

OK that's clearer now.

Is your protein a phosphate binding one? I can't see a reason why changing from 2mM to 10mM would change the binding characteristics otherwise. 2mM to 100mM or higher, yes, but not that small a change.

But if you're sticking to that protocol:
1) In no particular order; keep it cool; add protease inhibitors; 10% glycerol; non-ionic detergents (Triton, Tween etc.)

2) Yes, you can do this. All you're doing is changing the interaction of the protein with the column.

3) Perhaps you could try changing the buffer from phosphate to (eg.) MOPS to see if the purification is better. You couldn't do the buffer strength change to elute your protein any more (MOPS won't go to pH8), but you will also not elute your 'other proteins'.

I'll post some more when I think of them.

DM

[nashnash]

Hi

Thanks Btw, your reply has made me curious about another thing - Is there any assay to find out if my protein binds phosphate? (I don't know for now whether my protein binds phosphate). And if its a phosphoprotein, is it advantageous to go for hydroxyapatite chromatography after ion exchange step?

[Safoolia]

Hi! I will be grateful for help with HILIC column adaptation for supernatant analysis. There is a mixture of proteins, peptides and dfrnt. methabolites in sample. I am interesting in polyketide analysis. Problem is to choose write buffer to make a column stable and robust during number of runs. Can you suggest something, please?

[DaveM]

nashnash,

The only tried-and-tested assay I know for phosphate uses malachite green as an indicator but it is relatively insensitive (low mM from memory) so may not be that useful. Invitrogen (and others) do advertise some kits which go lower in sensitivity.

Hydroxyapatite may work if the putative phosphates are clustered together. I'm afraid this is the point in the thread where the only answer is 'try it and see'. It's always easier to predict behaviour if you know what your protein is, or what it's doing.

DM