Are results obtained with Ni-NTA sensor chip in SPR reliable?

Hi.

I plan to analyze binding affinity with SPR (biacore).

I want to use NTA sensor chip because my ligand-protein (10 kD; no Lys residue)has 6xHis tag.

But the problem is that the analyte protein (70 kD) has 6xHis too.

So after ligand immobilization, I want to block the remained Ni-NTA functional group (which is not binding with ligand).

This is my step by step protocol for SPR analysis.

1. Activate NTA sensor chip with Ni.

2. Immobilize the His tagged ligand.

3. block the chip. ------> this is waht i want to know

4. inject the analyte with various concentration.

Actually, when I asked about it to GE healthcare technical support team of our city, She said How about change the sensor chip to CM5 and change the position of ligand and analyte.

Because of my 10 kD His tagged peptide has no Lys residue, I cannot immobilize it on CM5 chip with amine coupling reaction.

I asked about L-Histidine for blocking agent too, but she said "The interaction between monomer histidine and Ni-NTA is too weak to remain on the chip during the experiments".

So, I think how about use polyhistidine (sigma, MW 5,000~25,000) for blocking reagent.

But I wonder if the polyhistidine is used as a blocking reagent, the immobilized 10 kD His tagged ligand is remain or competition reaction with polyhistidine.

Is there anyone who did like me?

Thank you.

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