protein purification problem - (Jul/29/2007 )
I am new in the field of protein extraction and now I should start to study plant secondary metabolism; I should extract total protein from plants and add precursors to the protein samples and examine the products with HPLC. The problem is that I have lots of secondary metabolites allready present in my protein samples after extraction. I have tried to purify the proteins with desalting columns after extraction but that did not helped much; I still could see lots of flavonoids and other secondary metabolites in my control (unfeed) samples in HPLC. So does anyone know how I can pyrify the proteins from secondary compounds so that they will remain in a nature form? Can I simply use concentrating columns to dilute and then concentrate my samples or should I precipite them with acetone or saturated ammonium sulfate? Or use affinity columns, but I dont know if some enzymes in the metabolite pathway will bound and some go to the unbould fraction? I would be very gratefull if some one could advise me in this matter. Thanks.
You can use ultrafiltration centrifugation unit with MWCO greater than 5,000 Dalton.
I think you could try to apply TPP extraction method which allow you to get rid of lyophilic compounds like flavonoids and others and make enrichment of your total protein mixture
IMO ammonium sulphate alone is not a good idea, it will preserve enzymatic activity but I noticed few times that at high concentration of ammonium sulphate phenolic compounds tend to stick to proteins (due to hydrophobic interaction increased by high salt concentration).
You can try to use PVPP to remove flavonoids (you can find protocols in some papers).