I am using the glutathione sepharose 4B beads from GE. I had noticed that the capture efficiency does decrease with usage, though I have successfully regenerated the beads at least 20 times and I am still using them.
I regenerate using 2CV of 6M guanidine after each run and every 5 runs with 70% ethanol as well. I have also used 5CV of 1M NaCl and noticed elution of proteins. I perform these experiments using the gravity flow method with a glass column. I have noted that the beads do clump together. A gentle re-suspension using a pipette seems to be good at unclumping them and improve capture. Also the diameter of the column seems to be important. I have noticed that using 5ml of beads on a 1cm diameter column captures far less than 5ml of beads on a 2.5cm diameter column. Hope this helps!
