use the following procedure. 1) immediately after use pass freshly prepared cold reduce glutation 20mM , 1mM EDTA in 50mM tris ph8.0. 2-3 CV,
2) wash alternate with buffer 1 and buffer 2 (GE lifescience), 2 cv, 4-5 times, then 1M Nacl 2-3 cv 3) wash with pbs , 5 CV 4) wash with 1% triton, 3 CV 5) wash with PBS, 5CV 6) wash with 70 % alcohol 5 Cv 7) wash with DW 10 CV 8) store in 20% ethanol. dont use GuHCL or urea unless absolutely require . Try to regenerate the beads on the same day of use. Vikrant ------------------------------------------------------------------------------------------------------------------------ Junior Research Fellow (CSIR) Lab No. 101, Dr. Varma Lab Cancer Research Institute Advance Centre for Treatment, Research and Eduction in Cancer (ACTREC) Tata Memorial Hospital, Kharghar, Navi Mumbai-410210 ##################################################################### ________________________________ From: Mirek Cygler <[email protected]> To: [email protected] Sent: Tue, 2 November, 2010 9:16:37 PM Subject: [ccp4bb] Glutathione sepharose Hello, For various reasons we are frequently expressing proteins with a GST tag. The glutathione sepharose beads that we are using for affinity purification seem to be difficult to regenerate and we see much lower capacity when used the second time. We are following the manufacturer's instructions for regeneration but this not very effective process as opposed to NiNTA which can regenerate multiple times. Due to this, the purification of GST-tagged proteins is quite costly. Do you know of a GSH-sepharose that can be successfully regenerated several times? Any comments will be greatly appreciated. Mirek
