Testing for binding of mutant proteins to cell surface receptors - (Oct/01/2005 )

Hi,
I would like to test for binding of a mutant protein to its cognate cell surface receptor. One protocol for testing the binding of this protein to receptors says to incubate cells with radiolabeled synthetic protein and unlabelled competitors. Specifically, the radiochemical is 125-I. The protocol also says that recombinant protein (not synthetic protein) is biotinylated with NHS-LC-biotin (I presume this unlabelled protein is used to get the non-sepcific binding).

My goal is to make mutations in the protein and test for binding on the cell surface.

I have a few questions about this procedure:
1. Does synthetic protein have to be used for radiolabelling or can I make recombinant mutant protein and then radiolabel it by myself? If I have to get synthetic protein made, can anyone suggest a company that does this and that may possibly make synthetic mutants?

2. Are there any other radiolabels I can use for this procedure so that I may test out the binding of the mutants?

I would really appreciate any help.

Thanks,
H

Hi Helen,
I think you should be able to radiolabel proteins yourself (I 125 is pretty nasty stuff though). I've never done this personally but I'm sure someone here will have a protocol for it.

The other protocol you seemed to mention was biotinylating your proteins which would be a non-radioative way to detect binding. Pierce make various kits for labelling proteins (http://www.piercenet.com/products/browse.cfm?fldID=62480CE7-7A66-45E6-B123-D09F4E0F9371). I think the NHS-biotin was probably for labelling amine groups. Then you could use FITC/PE labelled
streptavdin to detect the binding by flow cytometry. It might not be as sensitive as the radioactive method.

I also know of one paper using this method to look at biotin-labelled TNF to wildtype and mutant forms of the TNF receptor. Todd I, Radford PM, Draper-Morgan KA, McIntosh R, Bainbridge S, Dickinson P, Jamhawi L, Sansaridis M, Huggins ML, Tighe PJ, Powell RJ. Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour.
Immunology. 2004 Sep;113(1):65-79.

You could also use the Biacore SPR technology if you have access to a Biacore machine and your pure protein preparations of receptor and ligand. This measures binding in real time and you can produce binding kinetics. It is expensive and if you haven't got access to the machine...

Hope that helps,
Ceri

HiYa Helen,

If you have recombinant protein, this is acceptable to use for radiolabeling BUT you must be able to obtain pure protein. Can you use a serum-free media to collect it (if from mammalian cells) or do you have a quick column chromatography method to clean it up??

I-125 is the standard for doing protein-protein interaction studies. This radioligand is not as nasty as other radioactive chemicals but note, some proteins are structurally modified by I-125.

An alternative to this is using flow cytometry with either a FITC-labeled antibody (direct) or non-labeled antibody that can then be bound by a FITC-conjugated antibody (non-direct labeling), against your ligand. Your only limitation here is that the mutations may lead to loss of antibody binding.

Hope this gives you some ideas.

AussieUSA.

Hi, Ceri and AussieUSA,
Thanks for your suggestions! I really appreciate your help!

Take care,
H