Hello Everyone,
the other day I tried to run a ellman's regent experiment using L-cysteine as my control. the overall objective was to plot the values obtained from the standards(L-cysteine) to generate a standard curve.Then Determine the experimental sample concentrations ( my actual protein that has 2 cysteines) from this curve.
Material:
• Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA
• 4.8mM L-cysteine Stock solution
• 10mM Ellman’s Reagent Solution ( in 1 mL of reaction buffer)
Protocol:
I Prepare a set of cysteine standards; these standards will be prepared by dilution the 4.8mM L-cysteine stock solution into final concentrations of 1.5mM, 1.25mM, 1.0mM, 0.75mM, 0.50mM, 0.25mM and 0mM of L-cysteine in the cuvettes. (Total volume of 800uL).
2. Prepare a set of test tubes, each containing 50µL of Ellman’s Reagent Solution and 500uL of Reaction Buffer.
3. Add 250µL of each standard or unknown to the separate test tubes prepared in step 2.
4. Mix and incubate at room temperature for 15 minutes.
5. Measure absorbance at 412nm.
Except, when I tried to my blank 0mM of L-cysteine( 50uL of ellman's regent, 750uL of reaction buffer). the spectrometer did not read anything at 412nm. it gave me xxxx values meaning something could not been read. The blank has 750uL of reaction buffer and 50ul of ellman's regent, (total vollume 800ul) there is no cysteine sample inside the blank. I think something went wrong with the ellman's regent concentration was too strong or weak for the spectrometer to read at 412nm. Or maybe the reaction buffer doesn't work well for ellman's regent. If anyone has any idea to make this ellman's regent protocol work or have another protocol that works. please let me know.
Thank you
Jesus A. Aldana
If you have everything in your sample except the cysteine, then this is your blank. Zero your spectrometer on this sample. Next, measure your other standards to make a standard curve. If possible, find a sample with a known cysteine concentration and analyze it as you would one of your samples. This will give you an additional control for your experiment.
Does your blank solution look colorless or yellow? It should be colorless. You should be able to set the spectrophotometer blank with this. If the solution is very yellow, then you have accidentally mixed cysteine into your blank, and the absorbance is probably too high for the instrument to measure.
Update. the Blank is 750ul of reaction buffer and 50uL of ellman's regent. The blank was also colorless. The samples were yellow, indicating the ellman's regent was reacting with the cysteines. The blank did not read at 412nm.
update. I already tried using a different spectrometer. I used the same spectrometer I used for my ellman's regent to blank and read a braford assay, it worked perfectly.
Can you verify the integrity of the reagent you purchased? If you cannot, order another small amount from another supplier. I have found that poor quality raw materials can sometimes cause problems. Just because it is fresh from a new bottle does not guarantee that is is not the source of your problem, and it's not something we typically think of when troubleshooting.
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