If you know the identity of ligands that help to maintain the integrity of the protein you wish to isolate, and you can supply these ligands, then you can include them during the purification to keep the protein stable. Otherwise, you can use generic stabilizers such as glycerol, sucrose or trehalose. You should also perform the purification at 4oC.

Typical protein purification procedures that are gentle include ammonium sulfate fractionation and various forms of column chromatography such as gel filtration, ion exchange, dye affinity chromatophraphy, and hydrophobic interaction chromatography. Affinity chromatography can be used if there is a way to immobilize a ligand of the protein on a resin.

In order to follow your protein through the purification, you should test each fraction for the presence and amount of the protein using a specific enzyme assay.

Because you are using plasma as the starting material, you may want to consider using methods to remove the albumin and immunoglobulins early in the purification. Together, these proteins make up most of the protein in plasma, so removing them will go a long way towards purifying your protein.

Thank very much professor for your extremely useful answers it was brought me to upper level of knowledge in purification methods of protein, apart from all these methods is there any dialysis method could be useful to me for purification of interest of protein?

Dialysis is not normally used for purification. It is normally used for buffer exchange, separating small molecules like salts and sugars from macromolecules. If your protein is very small (

Thank you very much dear Professor, Yes Professor, I read about ultrafiltration but i dont have much experience on it. So i left it. Also upon ultrafiltration i felt, the presser may be affect the structural integrity of protein. I used Vivaspin 6 from GE healthcare, it is not that much good while i am seeing the protein profile of retentant in native PAGE. I need a very miled condition like gel filtration chromatography by sieving effect with low pressure. Thank you very much for your kind reply. 

Your sincerely

sakthivel

Gel filtration (aka size exclusion chromatography) is very gentle as proteins are not bound by the matrix, but the amount of protein that can be loaded is quite small, and the flow rate needs to be low (~ 1 cm/h). Thus SEC is used typically late in a purification scheme (polishing phase).

In the initial extraction phase you stabilise the protein. Remember that there are a lot of proteases in plasma, that need to be inactivated and then separated from your target protein. You may also need to add cofactors, chelators, buffer etc. The extract may need clarification (in plasma if there is a lot of lipid present). You can also do crude methods like ammonium sulphate precipitation or, if your target protein is heat resistant, heat denaturation. You may also use ultrafiltration for concentration (e.g., Amicon cells, use a membrane pore size that either allows as many contaminants to pass, but retains your sample or vice versa).

This is followed by the capture phase, where you use high capacity, fast methods to concentrate your target protein. Typical methods at this stage are IEC or affinity (e.g., IMAC) with coarse media and step gradients at high flow rates (several 100 cm/h). Ideally, conditions are chosen so that most of the irrelevant proteins are removed with the flow-through and do not even bind to the column. Go for removal of junk rather than yield of target protein.

This is followed by purification sensu strictu. You remove impurities and concentrate the target. Use medium size beads (10-50 µm) , gradient elution, moderate flow rates (~ 100 cm/h). Make sure that the sequence of methods makes sense, for example, use HIC after IEC since HIC requires the sample to have high salt concentrations that are often present in the eluate of a IEC column. 

Last, you use polishing steps to remove traces of contaminants and to ensure that your product is in a buffer where it survives long-term storage. Use high resolution methods (fine beads of < 10 µm, low flow rates, shallow gradients). By this time your sample is valuable (you have invested a lot of work in it), so yield is more important than at earlier stages.

If you do not really understand what I have been talking about, read a book on biochemistry laboratory methods first. If I may say so myself, Buxbaum 2011 is not the worst choice, but there are others. Your university may also offer appropriate courses. An afternoon spend in the library can save you months of work in the lab (been there, done that). 

 Dear Professor (Engelbert Buxbaum)

        Thank you very much ! what an amazing answers from you, I really salute you Professor. Excellent answer for my question and very beautiful key notes to understand purification step strategies with fine way, after reading your answers I got clarified well and good.  This what my professor also suggested me to go through the way. I miss you lot.  Your answers have been attracting me a lot !. I hope you are doing well and good, and I wish you the same. Once again I thank you for your valuable time spending and writing text to me.

Your sincerely

Sakthivel