SlyD seems to be a problem when im purifying my membrane protein. Why is it binding so tightly and how can I prevent this?
I worked with SlyD protein from E. Coli and solved its structure by NMR. When I was purifying the sample I used two affinity steps. The first one was a NiNTA affinity step (SlyD has enough Histidine residues in its C-terminus to be able to bind to nickel resin ) and the second step was a DEAE column. SlyD binds to the DEAE column in buffer containing less than 100 mM NaCl (or KCl). So you could try to remove it from your sample with a DEAE column, hopefully your protein won't bind to the resin, as well.
Extracting membrane-bound proteins has everything to do with the buffers you are using, and often you will have to try a wide variety of buffers to find one that works for extracting your specifc protein of interest. Do a google search to get an idea of what different protocols are out there.
I worked with SlyD protein from E. Coli and solved its structure by NMR. When I was purifying the sample I used two affinity steps. The first one was a NiNTA affinity step (SlyD has enough Histidine residues in its C-terminus to be able to bind to nickel resin ) and the second step was a DEAE column. SlyD binds to the DEAE column in buffer containing less than 100 mM NaCl (or KCl). So you could try to remove it from your sample with a DEAE column, hopefully your protein won't bind to the resin, as well.
Thank you, that is very helpful. you think monoQ will work? I don't have DEAE column but I do have monoQ
Hi Joe,
Here's a recent article with an example of SlyD contaminant removal by affinity chromatography which could also be of assistance:
http://www.sciencedirect.com/science/article/pii/S1046592817301651
Hope this helps,
Eric
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