RameshSundar

Hi,
A Brief background about the work: We are studying Sugarcane x Colletotrichum falcatum (Fungus) interaction at various dimensions. A part of the study is to identify the elicitor molecule of the pathogen which can elicit defense upon perception by the host. We identified that the elicitor of C.falcatum is proteinaceous in nature. In order to isolate the elicitor, we have to fractionate the crude protein extract and screen the elicitor-active fraction by biological assays. Active fraction has to be purified further until isolation of single protein with intermittent biological assays for screening.
We have tried two methods in Biologic Low pressure Chromatography system (Biorad).

• Size exclusion using Sephacryl S200HR resin in 1x40cm econo column.
  
• II) Anion exchange chromatography in High Q cartridge supplied by Biorad.

In both these methods, We are not getting enough fractionation/separation; most of the proteins getting eluted in first two peaks itself and remaining few peaks did not have proteins; Also, the detector pointer did not reach to baseline between peaks which might be the reason for no fractionation. The student has done various troubleshooting steps to make the best out of it, but could not make it. When referring literatures, most of them used AKTA system with HiTrap columns for this purpose.
We do have HPLC with all types of reverse phase columns but no ion exchange columns.  We decided to buy a new ion exchange column specifically for the above purpose.
Seeking suggestions/guidance:

• What type of columns would be good to do both fractionation and purification? Will that be suitable for Biologic LP system or HPLC?
  
• Else do we need to change the strategy of purification?

Looking forward to hear from you with your valuable suggestions. Thanks for your time and support. With sincere regards….Ramesh