I have done dilution series of the total IgG standards 12 times, when I check the od readings from different dilution series (1:2; 1:3; 1:10) they give a sigma shape graph, but when I align with the concentration (500ng) going down, it does not align well hence i loose the shape.
The two graphs are attached.
Check the protocol for the kit you are using to see how they recommend fitting the standard. You may need to graph on a log scale or use a 4-parameter best (logistic) fit.
I do not understand: the ordinate should be the same in the two graphs: the absorbance (as in 2)
on 2; how concentration (abscissa) can it be zero?
send me your numbers
attached is the spread sheet with all the numbers. the plate has the standards in different dilution series,. my number as in?
You lose the shape in the second graph because the concentration values you are using for your x-axis are so small. Excel fits these numbers into the graph as they are, therefore all your concentration values cluster together around the y-axis (x-axis concentration near 0) and you lose your shape. It also doesn't help that you are comparing concentrations from 0 to 500. That is a very big range. You could try graphing the first few concentrations to see how the graph looks without the large concentration values and then go from there. It would require a bit of fiddling with the graph axis settings in Excel. Hope that makes sense.
Check the protocol for the kit you are using to see how they recommend fitting the standard. You may need to graph on a log scale or use a 4-parameter best (logistic) fit.
It seems that you are getting a sigmoidal curves in certain dilutions schemes (1000 ng, 1:3; 500 ng, 2:3; 500 and 100, both 1:10 are also sigmoidal). The dilutions schemes you use, are not clear, which are critical to get a sigmoidal curve. Usually, in an ELISA, you make serial 2-fold dilutions, about 8 to 10 dilutions that should cover the upper and lower asymptotes range, which give sigmoidal shape to ELISA curves.
When you are using a serial dilution, as in your upper curve, your X-axis is actually in a geometric progression. For example, in a 1:3 dilution series, the unit scale in the X-axis are in 3^(-1), 3^(-2), 3^(-3) and so on (i.e., log scale based on 3). So if you change your X-axis in the lower graph to log scale, or change your IgG concentrations to the log value based on 2, or 3, or 10 (whichever dilution series you want), the shape of the curve will match exactly to the upper curve but in mirror reverse. Remember, in the upper curve, your IgG concentration is higher on the left (lower dilution = higher concentration), in contrast to your lower curve, where the higher concentration is to the right.
Sorry for the late reply. You have acceptable calibration lines in some areas of concentrations (in yellow): Do not work with too high or too low concentrations
The answer is the same in dilution and in absorbance (see example from your data)
I find that variations in DO (or absorbance) is low.
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