My graph for standard curve doesn't touch at zero. I always get a graph straight but touching higher at about 2 parts. I am using the lowery method and the procedure is like this:
2% Na2co3 dissolved in .1N NaoH (A) mixed with 1% Nak Tartarate in .5% CuSo4.5H2O (B)
A(50ml)+B(1ml)=C
Folin reagent/Distilled water 1:1
.2,.4,.6,.8,1ml of BSA(50mg dissolved in 50ml DW) taken in test tubes, added to it 5ml of solution (C) then after 10 min I added .5ml of folin reagent prepared fresh. Then after 30 min we take reading at 660nm.
Please convey any suggestions if there is any fault in the process.
Thank you.
If you are referring to a standard curve with absorbance on the y-axis and protein amount on the x-axis, the intercept on the y-axis is the absorbance baseline when no protein is present. It is the absorbance of the reagent itself. It does not have to go through the origin.
As Adam stated, the origin does not have to go through zero due to the absorbance of the reagent itself, but also due to possible small contaminants in your vials, water, etc. Do you subtract out a 0.0 mg/ml BSA from all readings? Nonetheless, a zero origin for your standard curve will not effect results, provided your samples are suitably diluted in the same media. Personally, I like to include 0.0, 0.05, and 0.1 mg/ml BSA standard on my curves, which can significantly enhance your dynamic range.
I agree with Adam and Carl. That is your baseline absorbency - background of the reagent itself. That is the your zero and you can subtract that number from all your result.
Assuming you've measured and subtracted an appropriate blank, instrumental noise and random experimental error will contribute to a nonzero intercept.
thanks all of you. i also just want to know that absorbance or intercept value could be use as standard value
I think that Carl and Adam are very right. Well, in a more easy way, put your blank sample (containing no protein) in the instrument and put it to zero reading (blank reading). Then take the readings of your samples containing different concentrations of BSA, whereby all of your readings will automatically have blank subtracted. This way your graph will directly touch zero.
I do not understand why chromatographers use photometric Lowry's method.
Please, use the HPLC protein assay method, which appeared in
Hayakawa K, Yoshinaga T, Hirano M, Yoshikawa K, Katsumata N, Tanaka T, Nagamine T. Protein determination by high-performance gel-permeation chromatography: applications to human pancreatic juice, human bile and tissue homogenate. J Chromatogr B 754: 65-76, 2001.
and also in
Hayakawa K, Guo L, Li XK, Kimura H, Yoshinaga T, Katsumata N, Nagamine T, Tanaka T. High-performance liquid chromatographic protein determination. Curr Top Anal Chem 2: 195-200, 2001.
if you subtract blank absorption from all readings , instrumental noise and random experimental error can contribute to a nonzero intercept
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