Q: Does this antibody also detect 8-OHdG?
A: The primary antibody used in our 8-OHG kit is monoclonal and will also react with 8-OHdG. We recommend using purified RNA samples to specifically detect 8-OHG. If using urine, serum or cerebrospinal fluid samples, both 8-OHdG and 8-OHG will be detected.
Q: How much RNA do I need to use?
A: The protocol calls for adding 50 µL of RNA at 1-5mg/ml, which is between 50-250 µg total. It is fine if your RNA is not this concentrated as long as you add a minimum of 6 µg/well.
Q: What nuclease P1 and alkaline phosphatase do you recommend?
A: We recommend nuclease P1 from Sigma #N8630 and alkaline phosphatase from Sigma #P5931.
Q: In what buffer do I need to prepare the nuclease P1?
A: Here is the buffer that we use to reconstitute our nuclease P1: 30 mM Sodium Acetate, pH 5.2 and 5 mM Zinc Chloride.
Q: What dilutions should I make of my plasma samples?
A: Plasma samples are not recommended for use with 8-OHG ELISA because the antibody used in this kit cannot distinguish between 8OHG and 8OHdG. The only way to determine 8OHG levels is to use a purified RNA sample. If the detection of both 8OHG and 8OHdG is not a concern, you can use diluted plasma samples. We recommend performing a sample titration prior to running the experiment to determine the necessary dilution for the sample to fall within the standard curve.
Q: Is this kit suitable for use with bacterial cells?
A: Since this kit uses isolated RNA samples, it is suitable for use with RNA extracted from bacteria.
Q: How do I make the standard curve and calculate my results?
A: The best way to determine concentrations from a standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel. When graphing the standard curve, set the x-axis to logarithmic scale and generate a linear or logarithmic trendline. The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within this range. The middle part of the standard curve is the most sensitive and is the best part to use for quantifying samples. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is (OD value-b)/m for linear lines or ln(x)=(OD value-b)/m for logarithmic lines.