I'm building an ELISA from an antibody pair kit (R&D Duoset DY291) and it seems I can't use BSA in case there's any bovine IGF1 (100% similarity to human form) present so it suggests I use PBS with 5% tween which is fine.
However the product insert then says “The PBS -tween (5%) diluent must not be used to dilute the Detection Antibody or the Streptavidin-HRP".
My question is what buffer should I use for the Detector & strep-HRP? Can i use BSA here or will it potentially compete off the human IGF1?
Owen
Does the PBS-T supplied as diluent contain sodium azide as preservative? Azide deactivates HRP. Some freshly prepared PBS-T (without azide) should do well.
I agree with Wolfgang. Tween itself does not affect the hrp enzyme, unless its high concentration. Normally, I used it at 2% not 5%. You can also use 1% fish gelatin instead of bsa in the pbs for your detection components. You can purchase it from sigma. If you are making batches of this and need a preservative that is hrp friendly, consider proclin 300. It is also a more effective preservatove than aside.
Lora Benoit Proclin 300 is hydrolyzed rather quickly, so it has disadvantages when a bottle is opened again and again for taking aliquots, while sodium azide will last "forever". For short term usage, one may omit preservatives completely. I like running alike solution through a bottletop filter, though, to remove particles and possibly microbes.
Thank you both for your prompt replys which are so useful with an ongoing troubleshooting problem! @Wolfgang I make my own PBS-T 5% so I know there's no sodium aside
@Lora i"m so glad someone else uses 5% successful!
For some reason my ODs are really poor (max signal:noise =2) and I'm very curious as to why the supplier says don't use 5% tween for detector antibody & streptavidin-HRP (see attached insert). The gelatin might be one way around this issue
Owen
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