Hello Everyone,
Can someone please tell me path length to be used in the calculations when 96 well plate is used for assaying the activity of enzymes! I assume we dont use 1cm path length as in microtiter plate the light passes vertically as opppsed to a cuvette!
Please correct me if I am wrong.
Thanks in advance.
Hi, Swati! The pathlength depends mostly on the total volume you use in your wells, and also the shape of the plate bottom and the meniscus position. Some readers can correct it as Jenny said, but you can assume that, for a standard flat bottom 96-wells plate, a volume of 350 microliters has a 1 cm pathlength. Other estimations are for a 100 microliters - 0.28 cm, 200 microliters - 0,56 cm, 250 microliters - 0,70 cm. If you want to check, you can measure the absorbance using a cuvette and compare with the absorbance obtained by the plate.
Well I think that the path length value won't matter in activity calculation, as the molar extinction coefficient value (slope of the standard curve) is also coming from the same path length.
Most microplate readers are capable of reading absorbance have a 1 cm path length correction built in via an internal fitting algorithm. Using this correction technique, the 1 cm data from a microplate assay is given with no calculations or estimations made by the user. The path length correction feature can be turned on or off via software as per your needs.
Hi, Swati! The pathlength depends mostly on the total volume you use in your wells, and also the shape of the plate bottom and the meniscus position. Some readers can correct it as Jenny said, but you can assume that, for a standard flat bottom 96-wells plate, a volume of 350 microliters has a 1 cm pathlength. Other estimations are for a 100 microliters - 0.28 cm, 200 microliters - 0,56 cm, 250 microliters - 0,70 cm. If you want to check, you can measure the absorbance using a cuvette and compare with the absorbance obtained by the plate.
Hi Swati, here is an empirical way:
fill 3 replicate wells with the same volume of water as your samples. Measure absorbance at 900 nm (A900) and 977 nm (A977). Calculate means for A900 and A977 and determine your pathlength (cm) as (A977-A900)/0.18.
The answers above have been of great help.
I am trying to standardize Corticosterone ELISA kit protocol, for which the manufacturers suggest us take OD readings at 405 nm and correction between 570 and 590 nm. In such case would it be accurate if I manually measured the OD for the buffer at 570 nm and 590 nm using a cuvette then calculated the K factor and pathlength correction for the microtitre plate according to the formula mentioned above Inna M Sokolova?
Please note the assay buffer provided is Tris-Buffered saline.
Hi Vidya, absorbance (A) at all wavelengths is proportional to the light path (L) (A=e * [C]*L), so as long as your K factor is the ratio of the OD at 570 and 590 nm (i.e. one divided by the other) and is measured at the same sample concentration as your OD at 405 nm, you can use it.
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