This is totally independent from the substance you are measuring, but strictly dependent on the color of the final product from your enzymatic reaction. If you are doing an ELISA, you are supposed to use a molecule conjugated to an enzyme, are you using that? If so, you should also use a substrate mixture to obtain a colour product, revealing the presence of the enzyme at the end of the assay. Depending on the composition of the substrate mixture, you can obtain different products and should use different wavelenghts,which are clearly defined.
For instance, if you use a molecule conjugated to the enzyme horseradish peroxidase, you can use at least two different substrates- OPD and TMB. In the first case the product is orange/red and read at 490nm. If using the same enzyme, but with TMB, the product is blue, changes to yellow after adding acid as described, and should be read at 450 nm. If you are using alkaline phosphatase as enzyme, the conditions are different...
Which substarte and enzyme are you using? Or are you performing a different assay (not ELISA)?
typical ELISA uses TMB substrate. after addition of acid to stop the reaction, the plate can be read at 450 nm.
Before this I only used kits to analyse my sample. But now i wont be using kit since the substance that i'm quantifying has not been produce in type of kit. So i wonder if i were to do it manually, how can i determine the wavelength? What do you mean "typical ELISA" and what is TMB substrate. I did search for this term but none shown what i really want to know. I might had look in the wrong place. Could you please guide me?
This is totally independent from the substance you are measuring, but strictly dependent on the color of the final product from your enzymatic reaction. If you are doing an ELISA, you are supposed to use a molecule conjugated to an enzyme, are you using that? If so, you should also use a substrate mixture to obtain a colour product, revealing the presence of the enzyme at the end of the assay. Depending on the composition of the substrate mixture, you can obtain different products and should use different wavelenghts,which are clearly defined.
For instance, if you use a molecule conjugated to the enzyme horseradish peroxidase, you can use at least two different substrates- OPD and TMB. In the first case the product is orange/red and read at 490nm. If using the same enzyme, but with TMB, the product is blue, changes to yellow after adding acid as described, and should be read at 450 nm. If you are using alkaline phosphatase as enzyme, the conditions are different...
Which substarte and enzyme are you using? Or are you performing a different assay (not ELISA)?
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