Hello everyone,
today I ran my ellman's reagent twice using a 96 well plate and tried to read the concentrations of my standards of my L-cysteine using a elisa plate reader and reading the absorbance at 412nm. Each well contained 50uL of ellman's reagent, 50uL of different concetrations of L-cystenine standards, and 100ul of sodium phosphate pH 8.0, total volume of each well was 200 ul. (I ran these standards in triplicates) The different concentrations of L-cysteine were 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.7 ,0.08,0.09, and 0.10 mM. I did a Bradford assay to check my L-cysteine standards concentrations before I ran them in my ellman's reagent to make sure the correct concentrations were being used. When I ran my ellman's reagent the absorbance did not make a standard curve and in addition, when I checked the concentration of my L-cysteine standards using beer's law they did not match up with the concentration of my standards. I originally did this experiment using cuvettes ( 500uL sodium phosphate pH 8.0, 50 ul of ellman's reagent and 250ul of each different L-cysteine standard). those readings made a standard curve and when i checked their concentrations using beer's law they matched up with my standards. I don't understand what could have gone wrong. The reason why i want to use 96 well plate for ellman's reagent is because it requires less of protein of interest and the concentration of my protein of interest will be higher in 200ul instead of 800ul. If anyone has any suggestions on how to fix my problem to use ellman's reagent in a 96 well plate and elisa plate reader or has a protocol that successfully works please let me know.
Thank you
Jesus Aldana