- His-tagged protein immobilized a on Ni coated NTA chip.
- Both the reference channel and ligand bound channel shows binding with analyte (ribosome)
- negative RU in the resultant curve.
- Reference channel is Ni free.
- Running buffer contains hepes,NaCl,EDTA,Mg(OAc)2
- Both the ligand and analyte are prepared in the running buffer.
- Reference channel was tried to be coated with BSA and still it's showing non-specific binding.
- Tris buffer as running nuffer gave the same non-specific binding but the resultant curve gave positive RU. As suggested in some paper I am using Hepes Buffer now. If anyone has any idea please suggest me a way.
- I have coated the reference channel with GFP still it's showing non specific binding.
- I tried to repeat the first experiment where i used tris buffer as running buffer and got some positive RU value in resultant curve but now it's not working.
if anyone come up with some suggestion it will be appreciated.
Try to improve the sample's flux in order to avoid these non-specific bindings.
Would you be willing to try a different sensor chip? perhaps a CM5 chip modified using NHS/EDC and then inject your protein sample to couple (via aminocoupling) it with the functionalized chip. But in this case, Histidine tagging will be irrelevant.
You could try increasing the salt concentration or adjusting the pH if it is a charge based non-specific interaction. If you can get the pH above the pI of the ribosome the non-specific binding might be eliminated. You could also try adding up to 1% BSA and up to 0.05% tween20 in the running buffer. Are you blocking the reference channel with his-tagged GFP?
We just posted an article on this subject. You can download the full PDF at the link provided.
http://www.nicoyalife.com/2015/11/23/spr-tips-4-ways-reduce-non-specific-binding-surface-plasmon-resonance-experiments/
Hi Sayan Bhakta, did you find out a solution? I have the same problem.
Dear Mariana J. Do Amaral, it was an experiment I was trying to do a long time ago. Unfortunately, I had to move on to other experiments as it was taking a long time to standardize. However, the main problem I was facing was the magnesium ions in my running buffer as far as I can remember. Magnesium being a divalent cation was probably binding to the NTA chip surface, giving negative RU. I also had cobalt ion in the buffer containing the his-tagged protein. Besides, ribosomes being such a big molecule needed more standardization which I didn't pursue. However, as far as I can remember, all the above suggestions from other people help understand the crucial points one needs to follow in order to standardize the experiment which I left inbetween.
- Badges
- Science topic
- Similar topics
- Indexes