Hi, I think that the following publication by Xiaolin Wu et al. will be very helpful:

https://www.nature.com/articles/nprot.2014.022

Dear Ghazaleh Abedi Sir,

Thank you very much.

That depends on what you want to use them for. If you want some functional characterization, than you need to use mild conditions and the conditions will differ from case to case. If you want just proteomic analysis and you don't care about the native state, than harsh conditions will be suitable for you as suggested by Ghazaleh.

Dear Tomáš Hluska sir,

I want to separate proteins with there functional properties as I have to mix them with something interesting.

Maybe if you were more specific, it could help. But as I understood, you want to test binding of some LMW compound to enzymes? One class, or several various classes? (e.g. only to different glucosyltransferases, or also to other enzymes?).

Anyway, if you want to test binding of anything, you must treat your proteins carefully. Keep the extract at 4°C all the time. Try buffers as Tris, Bis-Tris or K-phosphate, pH 6.0 - 8.5 (but more acidic pH may be more suitable for some proteins). Try some salts, KCl at concentrations from 50 mM to 2 M. Try addition of glycerol (5%), maybe some detergent (Triton X-100, Tween 20, dodecylmaltoside; each of them requires different concentration).

You can get some idea here:

https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/solubility_studies/

It is for extraction from bacteria, but you can get the idea what you can try.

Since you need a functional protein, here is what I would suggest. Try making extracts with a few different buffers - at least one between pH 5 and 6 using a citrate-based buffer, one around pH 7 or 7.5 using a Tris-based buffer, maybe also one of each with added dithiothreitol in case you need to keep sulfhydryl groups reduced. If you think your protein of interest is membrane-bound, then use detergent like Triton X-100. If not, I would omit detergent. Then, use the extract to test for activity. If you find at least some activity, you can try to enhance the activity by making some changes, perhaps adding 10 mM NaCl, switching buffer ion and so on. Only then would I suggest trying to purify. For purification, I find using ion exchange spin columns to be useful for testing out conditions. The columns are small and chromatography is done by spinning in a microfuge. You will want to choose a matrix and condition where all your activity is bound, then try different elution conditions. The aim is to get most of your protein of interest in the smallest number of elutions, while removing proteins that you don't need. You would keep an accounting ledger noting down extract volume, activity per unit volume and mg protein per unit volume, so that you can select the best procedure and refine as necessary. Alternatively, if you are looking for a protein that binds a specific compound, instead of ion exchange, you might want to make an affinity column with that compound.