Oct 21, 2021
Luminol Calibration
- 1Philipps-University Marburg
Protocol Citation: Michael Burgis 2021. Luminol Calibration. protocols.io https://dx.doi.org/10.17504/protocols.io.bzdfp23n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54407
Abstract
Calibration curve using luminol in order to standardize luminescence data
Preparation of the Buffers
Preparation of the Buffers
Preparation of 100ml luminol stock solution (1 mM luminol sodium salt; 50 mM sodium carbonate; 300 mM sodium bicarbonate; 5 mM ammonium carbonate) :
dissolve the following components in ddH2O
19,94mg of Luminol sodium salt
529,94mg of sodium carbonate
2,52g of sodium bicarbonate
48mg of ammonium carbonate
Preparation of the coppersulfate solution (1,5 mM CuSO4):
dissolve 23,94mg of CuSO4 in ddH2O
Preparation of dilution series
Preparation of dilution series
Perform a dilution series in a 384 well plate similar to the protocol provided by the iGEM foundation on Fluorescein calibration:
Add 100µl of the CuSO4 solution in the first well
Fill every second well in that row with 50µl of water (8 rows in total)
Transfer 50µl of the first well into the next and carefully pipette up and down to ensure proper mixing of the solutions
Repeat this until you reach the second last well and then dispose the last 50µl
Perform the measurement
Perform the measurement
Premix the luminol buffer with hydrogen peroxide in a ratio of 4:1
Transfer 10µl of this solution into empty wells on the plate
Use a multichannel pipette to add the inducing copper sulfate solution to the luminol solution
Transfer the well plate into the plate reader and shake the plate for 30 seconds until you start the measurement
Measure the luminescence with an exposure time of 250ms
Plot the data in a linear correlation and use this calibration curve in order to normalize your luminescence data