Q: Which detection method is more sensitive?
A: In our hands, the chemiluminescent and colorimetric formats display similar sensitivity. In general, luminescence usually offers better sensitivity and a broader range, and the chemiluminescent assay format may be more sensitive with certain luminometers.
Q: What is the recipe of the lysis buffer?
A: The lysis buffer included with the kit contains 50 mM Tris, 0.1% BSA, 2% Triton X-100, 0.01% Thimerosal, pH 6.0.
Q: Why is the lysis buffer provided with the kit interfering with my protein assay?
A: The lysis buffer that is provided with kit contains BSA, which will interfere with protein assays. If you plan to perform a protein assay, you may prepare your own lysis buffer without BSA using the following recipe: 50 mM Tris, 2% Triton X-100, 0.01% Thimerosal (optional), pH 6.0.
Q: Why is there an optional acetylation protocol?
A: Sample acetylation can increase the sensitivity of the assay 10-fold because the antibody has a higher affinity for the acetylated form.
Q: How do I calculate my results?
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. When graphing your standard curve you will want to set the x-axis to logarithmic scale and generate a logarithmic trendline. The standard curves generated with ELISAs are not typically linear, therefore the points of the curve that aren’t in the linear portion should be eliminated, so that you get r2 as close to 1 as possible, as long as your samples still fall in that 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 ln(x)=(OD value-b)/m for logarithmic trendlines.