I have a question about recombinant protein SPR experiments: After the recombinant protein was pre-enriched recently, the SPR test results were abnormal (such as weak signal/high non-specific binding).
What factors may be related? It is known that pre-enrichment uses affinity chromatography. I would like to know whether there is room for optimization from protein purity, concentration to buffer compatibility.
I hope to share your experience! Thank you!
[Troubleshooting Questions are selected from MCE customer consultation emails.]
When using the CM5 chip for amino coupling, the following points should be noted:
Protein preparation ingredients:
It is necessary to confirm whether the recombinant protein contains Tris in lyophilized form, which may interfere with the carboxyl activation process on the chip surface and affect the protein coupling efficiency.
Charge matching conditions:
The buffer pH value should be controlled in the range of > 3.5 and < the isoelectric point (pI) of the ligand protein to ensure that the protein carries a positive charge and forms electrostatic adsorption with the negatively charged surface of the CM5 chip;
At the same time, the protein pI>4 is required to meet the basic charge conditions of amino coupling.
If the above conditions are not met, the amino coupling method may not be applicable to the recombinant protein, and it is recommended to use a capture method (such as antibody capture or His tag capture) instead.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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