Here's a set of opinions from a LOT of experience, not all good:

Tris should be avoided. It is very temperature sensitive, an amine, and is a good general base catalyst.

For any buffer, you need to maintain control of ionic strength as well. Thus, two buffers at (nominally) pH 7.5 could have VERY different ionic strengths.

And, here I can help you out. Many years ago I wrote a calculator for construction of thermodynamically corrected buffer recipes. It has now been used to prepare three quarters of a million litres of buffers. I am aiming for a Megalitre before I retire!

Link:

http://www.liv.ac.uk/buffers/

Hi, have you tried a stability assay using thermofluor technique?

You can find more info in www.thermofluor.org

@Sara Zamora: I guess you need purified protein for that, don't you? Nada Mohamad will obviously purify the protein in future.

@Nada Mohamad: I doubt anybody will be able to answer only from these numbers. Many proteins may have such properties and yet require different buffers. It also depends on what they do, what amino acids they have in active site, etc.

I'm currently solving the same problem, unfortunately I get different results every week :-/ There is probably no other way than to extract in different buffers and measure the activity after some time.

A good point to begin would be to use common protein purification buffers like Tris or Sodium phosphate. You may have to consider the following:

1) The proposed purification procedure

2) Temperature at which you intend to carry out the process

3) Source of the protein? Mammalian, bacterial etc

You may start with buffer at pH-8 (Tris, NaH2PO4, NaCl) in which most protein those even with high PI (9-10) acn be purified. If the binding efficiency is very poor, you may reduce the pH range (5-6) which may improve binding...

Tris -buffer

Thank you all for your answers. i tried buffers with Tris ph 7.5 , Nacl, Glycerol etc.. ) but am not getting the optimum results i want !! and i want the maximum amount of soluble proteins to crystallize later on.

What's optimum result for you? You may spend years with optimalization and getting nowhere actually, so if it's working, better take that and be happy that your enzyme lasts at least few days ;) Personal experience.

Nada,

please define optimum?

If you get some soluble protein with your protocol count yourself as lucky. Of course you can spend time in optimization of purification, and you should, but I'd recommend purity/homogeneity over yield any time if you intend to crystallize it.

Affinity chromatography followed by ion exchange and size exclusion should be the minimum. If you loose 80% along the way, that is OK. Just grow more cells the next time. If you have a good homology model you need only one (1) good crystal.

The additives (salts (what salt?), glycerol, reducing agents etc.) may or may not be necessary. That also depends on the enzyme and needs to be examined.

Yes am getting protein from both with the buffers i tried but a very little amount although i´m increasing the scale of culture i´m using but one of my proteins is aggregating after size exclusion chromatography and the other is degrading some how.

What proteins are they? (oxidoreductase, transferase, hydrolase...)

What additives do you use?

Nada,

1) try to optimize the lysis buffer first. The more protein you can initially capture the better. It is also the easiest experiment to do. 2-4 gels and you are done. Try 50mM buffer (pH 5.0 to 9.0), 20mM DTT (overkill DTT rarely hurts but often helps ), 50 to 1000mM NaCl and 0% to 20% Glycerol first. Also try adding ligands to the lysis buffer if you can. High salt or glycerol CAN precipitate some proteins so always cover the lower end.

2) If 1. does not help go back to the expression. What cells do you use, the temperature, the media, how harsh you induce over expression.... You will be surprised how many purification problems can be solved by fine tuning the expression. Also check and modify you expression construct. see 3.

3) Protein degradation can be good. Does it form a stable higher molecular weight species? Try to verify that with limited proteolysis and Mass-Spec. Clone the stable fragment if it serves your purposes. The fragment might be still active in-vitro. You can try limited proteolysis for your aggregates, too.

4) Some people use L-Arginine or L-Glutamate to de-aggregate proteins.

5) Forget about those sequence based pi values when choosing a buffer, especially when you have dimers, multimers, aggregates or complexes.

6) If nothing works, try refolding.

good luck.

I had to do something really bad in my past life, because DTT killed my enzyme and Arg+Glu did not work for me :( Also putative ligands decreased activity (but that may be rather due to inhibition).

Here's a set of opinions from a LOT of experience, not all good:

Tris should be avoided. It is very temperature sensitive, an amine, and is a good general base catalyst.

For any buffer, you need to maintain control of ionic strength as well. Thus, two buffers at (nominally) pH 7.5 could have VERY different ionic strengths.

And, here I can help you out. Many years ago I wrote a calculator for construction of thermodynamically corrected buffer recipes. It has now been used to prepare three quarters of a million litres of buffers. I am aiming for a Megalitre before I retire!

Link:

http://www.liv.ac.uk/buffers/

Hi Nada,

It is difficult to advise unless we know the identity, sequence, and functions of the proteins.

As suggested above sometimes the only way is to test many buffer conditions and come up with something that works sufficiently well to yield enough protein (optimum conditions might take a lifetime!).

To simplify the search, first look for any published work on these proteins (presumably you have done that).

Secondly, check for homologues or other proteins that belong to the same family of proteins or similar proteins from other organisms. Copy the purification procedures for these.

If none of the above yield anything useful, then start with standard buffers (Phosphate buffers work for proteins of a wide variety) and work from that.

There are some great books on protein purification.

A good one to start with is the CSHL press, Basic Methods in Protein Purification and Analysis: A Laboratory Manual. ISBN 978-087969-867-6

Good luck!