I am in a process of separation of two proteins which have the difference of one aminoacid in the structure between them. I am looking out for a RP-HPLC method. Can any body help me out in separation of proteins structurally related very closely...with mobile phase & stationary phase conditions.

regards,

The column choice as well as LC conditions depend on the size and the structure of your proteins. Several companies including Agilent, Vydac and others propose various columns that are specially dedicated to proteins separation (C3 or C18 with a large pore diameter 300 A). The main objective is to exploit the nature of this amino acid which differenciate your proteins.

The Molecular Wt. of the protein is around 5 kD and I am looking for the separation in a preparative scale. what type of buffers can I use generally to separate these type of typical compounds and what pH shall I adopt.

Dear Anonymous, what do you mean with preparative scale? We are producing process resins and pepacked columns for analytical and preparative use, as some other manufacturers. Perhaps a look into our Resin Finder and/or Column selection guide, available on our webside www.tosohbioscience.com will give you an idea of how you can start. Good luck. Gerhard

What are the two amino acids that are different between the different proteins? If they have a basic or acidic side function, you may have a shot at separating the proteins with pH manipulations. If they are both neutral amino acids in the middle of the chain, your chances for a separation are rather slim, but not impossible.

Now I am very confident and further to continue my discussion it is the separation between the Recombinant Human Insulin and Desthreo, I am looking for process scale reverse phase chromatographic method, any body please let me know the conditions of the polishing step (final purification).

Insulin is a rather small molecule that is best separated on a 10 nm column. You also will have a higher loading capacity on a 10 nm column compared to a 30 nm column. I recommend The Atlantis dC18 column for this application. In a comparison of different column chemistries for peptide separations it has shown the best peak capacity. You can have a look at a representative separation of small proteins at http://www.waters.com/watersdivision/waters_website/pdfs/WA20701.pdf. I recommend to start of with 0.1 % TFA in your gradient, since you ultimately want to do prep. Even a higher concentration is possible, if needed.

Looking for a final polishing step my recommendation is a polymeric RPC resin. Benefits for such a resin are it's chemically resistent for CIP and sanitization using up to 1,0M NaOH, long life time etc.! Effect of molecular size and loading velocity on kinetic capacity to 1% breakthrough with 5mg/ml Insulin is described in our literature. Process methods and conditions of the polishing step are very sensitive issues and should be discussed between you and our process experts. They also can give you assistance in packing process-scale columns. Please contact my colleague in the US at Al.Jackewitz@tosohbioscience.com! Good luck. Gerhard