Hi all,
I just tried using a pre-coated ELISA plate for the first time, and I have a question about the results. I started by doing a dilution curve for two samples to see what would be the best dilution for my target protein. The absorbance of all the technical replicates are very consistent, and after I used the std. curve to calculate the concentrations they are still consistent. However, after multiplying the concentrations I got by their dilution factors, I got the highest concentrations in the highest dilution, with the concentrations decreasing as the dilution decreased. I added a photo to show what I mean. What could be happening here? It seems like a technical error but I don't know what the source would be.
Thanks.
To provide a better answer, we need to see the data, because you would have one of the following problems.
You dont have linearity in your std curve, or your samples are out of the curve, and for that reason your equation gives bad interpolations.
Your sample or diluent has an interfering substance.
Or you have a high background from the plates. What is the reading when you have no sample added, just buffer? Because if you have a reading of 0.11 when you make a 100-fold dilution, that seems to me it is the background value.
Michael J. Benedik Sorry, I just realized that photo was unclear. Those are my calculated concentrations (ng/ml) from the OD reading (not showing the readings themselves). For the 100 fold dilution the OD was negative after subtracting the blank, so clearly that one needs to be thrown out anyway. But the 10 fold dilution one is still confusing to me.
It may be that you are in a non-linear range, or at least partly, which might explain the anomalies.
If you have sufficient sample, I would try doing something like a 2-fold dilution series and read the raw numbers from the machine (along with the calculated numbers, and maybe by comparing them you can see where things are going wrong.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue