Coupling of protein to NHS activated HP column (GE healthcare)? - (Mar/05/2010 )
Ive read the protocol and I looked it up online but im no biochemist so its not obvious to me what would react with what and why. Could someone explain the chemistry behind the coupling reaction? I know that my protein is covalently linked to the matrix in the end and I have to wash with buffer this and that to get rid of unspecific binding and deactive excess active groups, etc but Id like to know exactly what im doing if possible
Comments or links would be awesome
Have a nice weekend!
how elementary do you want the response to be?
nhs binds to primary amines. proteins are made of chains of amino acids. one terminal amino acid will have a free amine group (primary in most amino acids). this will covalently bind to the nhs which is attached to the stationary phase.
you then want to block any unreacted nhs groups and wash.
Here's a basic animation of what's going on in there by GE:
This is a great place to start, it's a link to the PDF for GE's Affinity Chromatography Handbook
Start with the first chapter, "Common Terms" and go from there.
For an in-depth, and most importantly, digestible chemistry explanation with helpful illustrations, get your hands on a copy of "Lehninger Principles of Biochemistry" by Nelson and Cox, Chapter 3.1 - 3.3 on Amino Acids, Proteins and Peptides, and Working with Proteins. That chapter is pertinent to chromatography theory and more. I have the 4th edition of this book and it's been a great addition to my library. I'm no biochemist either, but I play one at work.
Haha thank you for the tips and info!! I really like reading about this stuff
I know about the amines binding to the NHS. They say something in the manual about isopropanol keeping it activated. I assume that ethanol amine binds to the free NHS but what about the pH changes in the buffers for the washing? The high salt removes unspecific binding but why add a pH4 step which shouldnt be great for proteins I guess?
pH 4, especially for short periods, won't necessarily harm proteins.
high and low pH washes help release non-specific binding to the matrix by changing the charge of the proteins.
protein that is covalently bound won't be released.