Q: Can this kit be used on samples from any species?
A: Because the structure of 3-nitrotyrosine (3-NT) is the same regardless where the protein lysate sample is from, STA-305 is not species specific and can be used for any protein samples containing 3-NT.
Q: Can I use serum or plasma samples with this assay?
A: You can use either serum or plasma samples. These samples can be added directly to the plate, undiluted, since this kit has a broad 3-NT standard curve that encompasses the 3-NT levels in most serum and plasma samples. If you find that the values fall outside the range of the standard curve, dilutions can be prepared using the Assay Diluent that comes with the kit.
Q: Can I use any lysis buffer to prepare my samples?
A: There is no restriction on the lysis buffer that can be used for cell or tissue lysate preparation with our Nitrotyrosine ELISA Kit. Detergent based lysis buffers such as RIPA buffer are compatible with this assay. Here is the lysis buffer recipe that we use:
* Tris 50mM, pH 7.5
* NaCl 150 mM
* NP40 1%
* Protease inhibitors such as PMSF or cocktail protease inhibitors
Q: Do I need to precipitate the proteins from serum samples?
A: No, serum samples can be added directly to the plate.
Q: What protein concentration should be used for this assay?
A: Our Nitrotyrosine ELISA is a competitive ELISA where a sample is added to a pre-coated plate, so the protein concentration of the sample is not critical. We don’t recommend a protein concentration for the Nitrotyrosine ELISA kit because the results will depend on the 3-NT levels in the sample. The kit has a broad 3-NT standard curve (1.95 to 8000nM nitrotyrosine) that encompasses 3-NT levels in most samples, so it is likely the 3-NT levels will fall in the range of the standard curve. It is recommended to start with undiluted samples and if the values fall out of the standard curve range the samples can be diluted with the kit's Assay Diluent.
Q: Why does the protocol call for 50 µL when other ELISAs require a specific protein concentration?
A: The difference is the format of the ELISA. The Nitrotyrosine ELISA is a competitive ELISA where the sample is added to a pre-coated plate, so protein concentration of your sample is not as critical as with an indirect ELISA. Indirect ELISAs require coating the plate with the sample and maintaining a consistent protein concentration throughout each well is important to ensure that comparisons are similar. We don’t recommend a protein concentration for the Nitrotyrosine ELISA kit because the results will depend on the 3-NT levels in each sample.
Q: Is it necessary to dilute protein samples such as plasma?
A: This kit has a broad 3-NT standard curve (1.95 to 8000 nM) that encompasses 3-NT levels in most samples, so it is very unlikely the 3-NT levels will be out of the range of the standard curve. It is recommended to start with undiluted samples and if the values fall out of the standard curve range the samples can be diluted with the kit's Assay Diluent.
Q: Why does the standard curve appear to be a reverse curve? Don't higher OD values correlate with higher nitrotyrosine levels?
A: The nitrotyrosine ELISA kit (STA-305) is a competitive ELISA where protein samples are added to a plate coated with nitrated BSA. The sample competes with the protein bound to the plate for antibody binding; therefore it generates a reverse curve. Low 3-NT levels in the sample results in more antibody binding to the plate and therefore a higher signal.
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 your standard curve you will want to set the x-axis to logarithmic scale and generate a linear trendline rather than a polynomial. 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 your 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 your samples. 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: If my sample falls outside of the standard curve, how do I adjust the volume?
A: If your sample values are at the high end of the standard curve, you will want to dilute your samples in the provided Assay Diluent while maintaining the original volume of 50 µL of sample that you add to the well. If your samples fall at the low end of the curve or at background levels, this means that the levels of nitrotyrosine in the samples are below the detection limit of this kit.
Q: How were the antibodies made and what was the immunogen?
A: The Nitrotyrosine ELISA kit uses a rabbit polyclonal antibody that was made using a KLH-nitrotyrosine conjugate and recognizes both free and bound 3-NT.
Q: Does the antibody detect free nitrotyrosine?
A: The antibody used in this ELISA can react with both free and bound nitrotyrosine; therefore this kit can be used to measure either free 3-NT or protein samples containing nitrotyrosine.