I am running a sandwich ELISA and realised far too late that I have run low on SigmaFast p-npp tablets for my detection step. In the product information insert, Sigma suggests 200 microlitres of p-npp per well. Can I get away with 100 without compromising my results or would I be best served to leave the plate in PBS over the weekend and wait until the replacement tablets arrive?
Dear Anne Marie,
You will do fine with 100 ul per well, but you cannot compare directly with previous measurements. The readings will just be somewhat less precise, but still very readable (the light path is only half the size but the concentration of enzyme is doubled, which theoretically should give the same result, although less precise).
Good luck.
Just
It's important: Volume meas thickness and that one of the factors in tge Lambert-Beer's law.
If you would like to increase your sensitivity, use 200 µL and double the concentration of your stopping solution and stop with only 50 µL. (e.i. 2 mol/L NaOH).
... but all background eaction will be increased as well.
In cases were coating antigens or antibodies are very expansive, we used a "saving" ELISA, working for coating and all incubation steps with 50 µL (and mild shaking) only, but with 200 µL substrate.
100 microlitres will do. As long as the tablets are in right ratio with the developing buffer.
I would guess that it s concentration rather than volume that matters. Naturally, you use 100ul and it is fine, all you need to bear in mind is that you need to embaptise your attached antigen into the buffers. So, I think it will be ok even with 100ul
Theodore is not right. It's not the concentration of the substrate. Substrate has to be always in excess so that the concentration (which is not the right term here, because of the enzyme is bound via the labelled antibody at the solid phase) or the number of enzyme molecules is the limiting part of this kind of reaction.
It's really simple physics: Lamert-Beer law: A = e b c
A - absorbance (no units, A = log10 P0 / P )
e - molar absorbtivity or extinction coefficient ( L mol-1 cm-1 )
b - path length of the sample ( cm )
c - concentration of the compound in solution ( mol L-1 )
Thickness doubled = Absorbance doubled.
Thanks so much everyone. It worked just fine with 100 uL. I knew it wouldn't be comparable to previous ones with more p-NPP, but wanted to be sure it wouldn't ruin my results.
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