I was looking for reconditioning method to reuse CM5 chips and I followed the protocol in Anal. Biochem. 229, p.112 -118 (1995) by Ronald C. Chatelier. However, I think it dosen't work for me.
In detail, when I measured RU of the bare CM5 chip, all flow cells were around 14200 RU. I used fc1,2, and 4 for immobilization of streptavidin or another proteins and they became to around 18000 or 16000 after I finished assays.
For reconditioning, I followed the protocol, except I used 1 mg/ml of PronaseE in 20 mM PBS (pH 7.4, containing 137 mM NaCl), not in "Sodium phosphate pH 7.2, 150 mM NaCl".
I don't think those two buffers are big differences. But anyway, I found that I failed reconditioning since RU of flow cells were still around 18000 or 16000 and I could see three imprints on the gold chip where the proteins were immobilized (fc 1,2,4).
Please give some comments on reconditioning of CM5 chips for me.
I urgently need you guys' idea.
Thank you in advance!
Not used this procedure often but it worked fairly well in the past.
Pronase E needs calcium, so you can try to add 3 mM and avoid EDTA.
Normally I incubate overnight on the bench in a humid chamber.
No other suggestions than try again and test the pronase E.
Thank you for suggestion!. I'll try adding CaCl2 for Pronase E incubation.
Do you have any other idea about reconditioning of CM5 chips? I've tried piranha solution like as cleaning gold surface. I expected to remove every organic compounds (even dextran) from gold surface and treated thiol-COOH chemicals for over night. After chip is equipped, I tested EDC/NHS-protein-ethanolamin immobilization. However, it showed far less RU for same amount of proteins to compare immobilization on new CM5 chip. Relative RU was about 400 after edc/nhs, 90 when 50ug/ml streptavidin was treated, and it became below 400 after ethanolamine blocking. Can I think the streptavidin is still immobilized on a chip in this case?
If yes, is this less binding capacity from the lack of dextran?
I look forward your answer , thank you in advance!
When you strip to the bare gold and build a new SAM the amount of binding spots is far less than with an dextran layer. If you compare a new CM5 (100 nm dextran) with a C1 (no dextran) you will see that you get only 10% immobilization compared to the CM5!
Not sure how to interpret your streptavidin immobilization (can you show a sensorgram?). But in the end I expect some binding. Did you try to capture an biotinylated protein?