cdc2kinase

I had a same problem and emailed GE customer center. Here is the reply I got (see below). Look like my protein is not stuck on the beads, it stuck on GST-tag and we can't do anything about it. I tried to elute with 4M UREA, but it was not successful. Only thing it worked partialy was eluting with 0.1% Sarcosyl like vivekp said.

"Thank you very much for contacting GE Healthcare with your question.

Unfortunately, your observation is not something easy to address—it needs more investigation.

Pretreatment of GSTrap column/glutathione sepharose resin does not help. The underlining issue comes from protein folding. GST-tag serves as a good carrier partner to keep fusion protein in solution. Yet, sometimes, the target portion of the fusion only stay soluble when interacting with the GST portion. And such interaction is so strong that even after proteolytic cleavage, thus two polypeptide chain, they still stay together in solution, as if cleavage did not occurred.  I am not aware of much consensus currently to address this problem. One of my colleague worked around it by screen out a few different construct, i.e. GST fusion protein with slightly different linker sequences. The only way forward, with your current construct, is to test out different conditions and, hopefully, one combination can be found to warrant separation. Additives such as detergent, low concentration of chaotropic agents (urea) are the normal direction. I would not consider SEC (size exclusion chromatography), rather ion exchange in the presence of urea. Anionic exchange chromatography (AIEX), such as mono Q is most widely used. Yet, please understand that as long as the interaction between target and GST is not fully disrupted, the complex will behave as one species in solution, thus chromatography separation is not possible."