I have acquired some NHS-activated HP columns (17-0716-01; GE), which are described for ‘easy coupling of ligands containing primary amines’. What does that actually mean? I am under the impression that all proteins contain amines, so does that mean these columns purify all proteins from a sample? e.g. if the sample was cell lysate, all the non-protein stuff will be lost, but the protein retained? Thanks in advance.
Follow up:
Assuming the column can be used to purify proteins which have exposed lysine, arginine or termini, and therefore not proteins without the amino acids lysine or arginine or that have buried termini. What percentage of proteins is that likely to purify? 50%? 80%? I don't see the point of the column if it only splits your sample into two pointless fractions. Am I missing something here?
NHS- coupled columns are more commonly used to immobilize an (already pure) small molecule, peptide or protein which are then in turn used to purify a protein from a mixture. The coupling chemistry is far too general to afford any specific purification from a complex mixture of proteins, many of which will present primary amines to the resin.
Usually a pure peptide or antibody is immobilized on the column, any remaining NHS is blocked, and the column is used to purify specific binding proteins. Often its peptide/antibody or antibody/protein binding pairs, with elution being with competing peptides, or more commonly low pH.
So, the column can be used to purify proteins which have exposed lysine, arginine or termini. Thus, proteins without the amino acids lysine or arginine, or that have buried termini, will not bind. Does that sound right?
NHS- coupled columns are more commonly used to immobilize an (already pure) small molecule, peptide or protein which are then in turn used to purify a protein from a mixture. The coupling chemistry is far too general to afford any specific purification from a complex mixture of proteins, many of which will present primary amines to the resin.
Usually a pure peptide or antibody is immobilized on the column, any remaining NHS is blocked, and the column is used to purify specific binding proteins. Often its peptide/antibody or antibody/protein binding pairs, with elution being with competing peptides, or more commonly low pH.
Note that the binding to the NHS column is covalent, i.e. non-reversible. As mentioned in the other comments, you cannot really use it to purify a protein directly; rather, you attach a protein which you can then use to purify some other protein (e.g. you can attach an antibody that is specific to your protein of interest to purify your protein from a cell lysate).
You will have to use pure (or nearly) pure protein for the coupling - if you use a cell lysate, all compounds containing primary amines will be coupled to your column, more or less in the same ratio as their abundance in the lysate. Therefore, your protein will most likely be out-competed by small molecules containing amines (e.g. free amino acids). Also note that when doing the coupling reaction, do not use a buffer containing primary amines (such as Tris), because this will couple to the column efficiently and prevent your protein from reacting with the matrix.
Thank you all. All good advice. So, in summary: I can use the column to bind e.g. an anti-venom (which is a collection of a few IgGs) or a toxin-specific IgG, as long as they are as pure as possible and contain no Tris or free amines. Then pass over my venom (collection of many toxins). The flow-through will contain toxins for which the anti-venom does not bind. The elution will contain the toxins which the anti-venom does bind. Bingo.
That sounds about right, Gareth! You might have to optimise your elution conditions, but a good place to start is a buffer at low pH (e.g. a glycine buffer at pH ~2), which should disrupt any antigen-antibody interactions.
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