Q: How long can I store my samples before testing for HNE adducts?
A: Free HNE is very unstable, but HNE adducts are very stable and samples can be stored at -80ºC for up to six months without HNE degradation. Freeze-thaws should be avoided.
Q: Are EDTA plasma samples compatible with your kits?
A: EDTA will not interfere with our HNE ELISA Kits and any anticoagulant can be used when preparing plasma samples.
Q: Is your HNE Adduct ELISA Kit compatible with samples of any species?
A: Our HNE Adduct Competitive ELISA Kit is not species specific and can be used with samples from any species.
Q: Is the HNE Adduct Competitive ELISA kit compatible with tissue lysates?
A: Tissue samples are compatible with this assay and lysates can be prepared in any lysis buffer.
Q: What cell number should I use in the ELISA?
A: Unfortunately we do not have a recommended cell number to use when preparing lysates, because this will depend on the HNE level of the sample, which will be different for each researcher. Our recommendation is to start with the most concentrated sample possible and prepare further dilutions later, if necessary, after running a small scale sample titration against the standard curve.
Q: Can PMSF and sodium orthovanadate be used in the lysis buffer?
A: PMSF is a protease inhibitor and sodium orthovanadate is a tyrosine phosphatase inhibitor and neither will interfere with any of our HNE Adduct ELISA Kits.
Q: How long will it take for the standards to become saturated when they are in substrate solution?
A: The time it takes to get to saturation can vary with each experiment, which is why we provide such a large range for the incubation time. Knowing when to add the stop solution is mainly based on experience and is an important step because if you wait too long the samples will be saturated. To avoid saturating the standards, it is best to focus mainly on the wells with the standard curve and you will want to see a bright blue color develop for the highest concentration and a very faint color in the lowest concentration. When you see a nice gradient of color you should add the stop solution.
It is best to add stop solution once a gradient has developed with the standard curve wells, rather than based on a set time point.
Q: How do I graph the standard curve?
A: The best way to determine concentrations from an ELISA 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. 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 equation of the linear trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is (OD value-b)/m.
Q: Can the 96-well plate be used over multiple experiments?
A: The HNE Adduct ELISA Kit comes with a strip well 96-well plate, which can be used as individual strips (8 wells/strip). Although the strips can be used individually, a standard curve must be run each time, which requires 16 wells.