Hi all,
Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC.
What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong.
thanks and regards,
meg
