Hi Alice,

There may be still some Imidazole left in the buffer exchanged solution, as you have eluted the protein solution with a very high conc. of imidazole.

Have you used any reducing agent in the buffer exchanged experiment?

In the buffer exchange experiment you can use 0.5mM TCEP and try to repeat the experiment.

Also, if you can't find any improvement, then you can try tagging the protein with Biotin. Then using SA chip you can surely have a good result in the SPR binding analysis

Good Luck !!

Also, you can have a look on our recent paper where we have also used a NTA sensor chip in the SPR experiment and found good binding of our protein.

Article Crafting of Neuroprotective Octapeptide from Taxol-Binding P...

Good Luck

Thank you very much Prasenjit for youe feedback... I also was thinking that maybe some imidazole was still in the buffer, so I performed two separate buffer exchange. Also, when eluting with flag peptide, the elution buffer was only HEPES and NaCl.

I've been only adding TCEP in the lysis buffer, not in the buffer I've been using for the experiment. Do you think iy would make a difference? when doing FLAG IP, I've been using DTT instead of TCEP as reducing agent in the lysis buffer though.

I've also been thinking of using GFP-tag since when I purify my protein using this tag, it comes out really clean....

Hi Alice,

I only use TCEP in the lysis buffer not in the buffer used for experiments. Haven't mentioned the concentration of TCEP you are using in the buffer exchange experiment, may be that was making a difference!!

Also you can try GFP-tagging of your sample but how you are going to rid off from imidazole.

Good Luck

Prasenjit

Hi,

Please add 1mM TCEP in your immbobilization buffer. Protein should be dessoilved into same buffer as well. Should work. All the best!

Regards