ELISA-increasing OD with greater dilutions - (Aug/29/2006 )
I've recently begun RNAi experiements in which I'm measuring the post-treatment level of the target protein by ELISA. The problem now (& not observed in sham controls) is that as I increase the dilution rate (from 1 or undiluted up to at least 1:64) of my samples, the OD also increases...instead of decreases as would be expected. I've never seen this before...has anyone ever experienced this? Any suggestions as to what may be happening would be most welcome.
What do you use to dilute (titrate) the samples?
I use PBS.
May be the reagent for titration is contaminated.
Sounds like you have some protein:protein interactions going on. As you dilute the protein, you go below a critical concentration and the complexes start to fall apart. Is the OD increase linear, or is there a definite concentration where it jumps suddenly?
Try diluting in different buffers.
I've recently begun RNAi experiements in which I'm measuring the post-treatment level of the target protein by ELISA. The problem now (& not observed in sham controls) is that as I increase the dilution rate (from 1 or undiluted up to at least 1:64) of my samples, the OD also increases...instead of decreases as would be expected. I've never seen this before...has anyone ever experienced this? Any suggestions as to what may be happening would be most welcome.
Sounds like you have some protein:protein interactions going on. As you dilute the protein, you go below a critical concentration and the complexes start to fall apart. Is the OD increase linear, or is there a definite concentration where it jumps suddenly?
Try diluting in different buffers.
if u ELISA not work ,u can check ur ELISA system,if ELISA work gud,check ur dilution buffer
gud luck
I think we have had something similar in cytokine/chemokine ELISAs in vivo samples (a peritoneal lavage fluid). We came up with two ideas about what was going on:-
1. We were diluting out an interferring agent in the lavage so the detected amount of cytokine was more when we corrected for the dilution.
or 2. The dilution buffer used was 1%BSA in PBS and that increasing the overall amount of protein in the sample helped the assay. Not sure exactly why that is but apparently it's a recognised phenomenon with ELISAs or luminex.
Maybe you could "spike" an aliquot of the sham sample with a mid-range dose of your protein standard and try your dilutions on this to see if you get the same effect.
All the best,
Ceri