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.
Hi Soi,
You could immoblise your analyte (70 kDa) to be your ligand on the chip as it has a his-tag or you could use a CM5 chip to immobilise your 70 kDa protein if it has lysines and then flow your 10 kDa protein as analyte for binding.
Just make sure you immobilise a lot of the 70 kDa protein on the sensor chip surface to amplify the signal when the 10 kDa analyte binds on the surface as the smaller the size of the analyte the more ligand needed to immobilised to get a decent response unit (RU) for binding. For the CM5 chip you block following immobilisation using 1 M ethanolamine hydrochlorid adjusted to pH 8.5.
Find the link for NTA chip and useful info:
https://www.sprpages.nl/sensor-chips-intro/biacore-sensor-chips/nta
Hope this helps.
Since your proteins both have His6 tags I am assuming they are cloned into an expression vector. In that case you may be able to simplify things by reengineering your constructs. Using PCR you could add a lysine residue to your protein to allow CM5 coupling. Alternatively, consider cloning your analyte protein into a vector that uses a different affinity tag or gives you a His6 tag that can be removed using a protease.
Perhaps, you can couple your protein to the chip through the amino terminal.
Bubacarr G Kaira Micah Jered Ferrell Jaime Guillén
Thank you for your reply.
I'm sorry to late.
I tried to use the methods that..
1. Immobilized the His-tqgged peptide as a ligand
2. block with poly-Histidine
3. inject analyte
I could get a binding sensorgram, however, the ligand was removed from the chip because of the regeneration solution (Gly-HCl 3.0 or EDTA included soln. or NaOH).
The immobilized His-tagged peptide was removed from the chip because of the regeneration soln., and whenever the different concentration of the analyte was injected, I had to immobilize the His-tagged peptide again.
It was a time-consuming method and it is difficult to maintain the orientation of the immobilized peptide.
So, We changed to used CM5 chip rather than NTA chip.
1. immobilize the peptide with amine coupling as a ligand
2. blocking with EA (mentioned above)
3. inject analyte
Thank you for your interest :)
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