Hi dear researchers,
I'm going to use ELISA to check a protein level in serum. I bought the ELISA plate coated with the antibody and I can get the protein concentration just by this plate.
What I want to know is, in my case, or any other commercial ELISA kits, is it necessary to have a positive control? I mean if I want to check the level of Protein A in serum, do I need to have pure Protein A (by protein induction and elution, mean I can generate in my lab) as a positive control to test the performance of the kit and at the same time as a reference to normalization?
Thanks for any reply!
Hello. I would highly recommend validating your ELISA for the samples you plan on using. You need to ensure that there isn't a matrix effect and that the kit is actually measuring what it should be measuring (tests of parallelism and standard addition). Pages 21 - 24 of the attached manual are incredibly useful for performing the tests of validation. After validation, it would be useful for you to have an internal positive control so that you can a) ensure that the kit is performing appropriately and b) calculate inter-assay variation. Good luck!
Yeah. As a matter of science in general -- if you have the opportunity to do extra tests to characterize your test, be grateful for that. It's never a bad idea to do that.
Hi Danielle and John,
Thanks to both of you! I know I need to do it now.
Yes, its better to always use.
Positive control, negative control and blank also
Add samples from E onwards on microtiter plate
Thanks Devendra!
I upload a Commasie blue gel and the band in red box is my aim protein. This lane is the elution from Ni-NTA beads. Because this is an overnight elute, can see below my aim protein, there are still some non-specific proteins, But since ELISA is a antigen-antibody reaction, is the quality of my elution ok to be the positive control? I mean although there are non-specific proteins remain, the antibody won''t recognize them.
Good experimental design is to use positive control & negative control....
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