I have a question regarding to your Amylose resin kit https://www.neb.com/products/e8021-amylose-resin
I read in the protocol https://international.neb.com/~/media/Catalog/All-Products/FF91B67397D945E0956F3483174CF7E3/Datacards%20or%20Manuals/manualE8200.pdf, that 200mM of NaCl is suitable for binding the MBP-tagged protein to the resin during the affinity chromatography, while during the separation of the r-protein from the MBP protein after the cleavage procedure, it was recommend to use a gradient of 50-500 mM NaCl to elute the protein and the MBP which elutes- according to the protocol- at 150-200 mM NaCl, so my question is: what is the effect of 200 mM NACL on the MBP-tagged protein binding to resin during the purification of the r-protein.
I am afraid that my MBP-tagged protein will be eluted with this high molarity of NaCl (200 mM).
I use the same pMAL protocol. I think the effect of the 200mM NaCl is to avoid any possible electrostatic interacion of other contaminating proteins to the resin, and that the binding occurs only by high affinity interactions (MBP-amilose). This high salt concentrations are common in other affinity purification such as Ni-NTA.
Yes, it does not make sense, with your initial conditions you should start the gradient at 200 and increase to 500 mM NaCl, if binding does not occur reduce the initial concentration of salt to 50 mM and see what happens. As Amanda says you are reducing the possibility of electrostatic interactions at high salt concentration.
I don´t really use a salt gradient for my protein elution with this method... I prefer using a pH gradient, and the results I´ve got are very good. You can try that if you know your protein´s pI. MBP´s is around 5.
Thanks Amanda,
I got you point , but in fact my protein in that case is a recombinant one, then the question is can I rely on the theoritical PI from the insilico work?
Thanks
Hi Omar ,
Thanks for your answer, this is the first time for me to use this protocol and your confirmation will encourage me just to go and try.
Thanks Omar
Hi Haitham,
My protein was also a recombinant one. I don't see why not to trust in the theoretical pI. It worked for me.
Good luck!
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