Hi everyone:
I followed the steps
known wt of tissue is homogenized with a certain volume of 0.1 N NaOH for at least 1\2 hour.
Then 1 ml of this homogenate was added to 5 ml of alkaline reagent solution in a clean test tube, mixed thoroughly and allowed to stand at room temperature at least 10 minutes.
0.5 ml of the diluted folin-Ciocalteau reagent(1:1 v\v) was added to above mixture and mixed immediately.
After 30 minutes, the extinction against appropriate blank was measured at 700 nm.
what is the formula applied to calculate % of proteins in sample?
The assays should always be compared to a calibration curve. One curve each time you restart an assay ! It is thus convenient to prepare several dilutions of the unknown sample and read them together with the calibration curve. Discard values outside the linear range of the curve.
You should prepare a calibration curve, with several dilutions of a standard protein (e.g. bovine serum albumine) and obtain the information about the linear part of the curve.
Pay attention to the fact that the calibration curve should be carried out simultaneously with the samples, otherwise you cannot be sure that the assay is valid.
Be also aware that the Lowry is a colorimetric assay subjected to reagent limitation and that the linear part almost always ranges from 0 to 0.5-0.6 absorbance units.
Do not hesitate if you need more information
thank you Dr./ Yannis for your reply
I already did that, I prepared CC and I got slope.
could you please tell me the next step? how to convert abs values into concentrations?
You divide the absobance value (Abs) by the slope :
example Abs = 0,100
slope = 0,005 (Abs/microgram per mL)
concentration of the sample = 0,100/0,005 = 20 micrograms per mL
then you have to take into account the dilution rate.
if the sample was diluted 2000 fold, the initial solution contains
2000 x 20 = 40000 micrograms per mL = 40 mg/mL
Step 1: You need to perform the experiment using different concentrations of a standard such as BSA (For eg. 1ug-10ug or in %age as in your case). Measure the absorbance and plot a regression curve with Concentration of the standard in the x-axis and absorbance in the y-axis.
Step 2: Plot the equation of a straight line (i.e. y= mx+c) using any statistical software or even MS excel.
Step 3: Put the absorbance value of your sample in the above equation. You will get the concentration of your sample
Hi there,
To make things simple, the calibration curve should make the relation between read OD and the total amount of protein present in the assay mixture (and not the concentration of protein). Then using this relation, you are able to determine the amount of protein present in any assay mixture provided the read OD is within your calibration curve. Then you can conclude about the protein concentration of your source of protein : it will be the amount of protein in the assay mixture divided by the volume of protein solution introduced in the mixture assay. If this protein solution is in fact a dilution then multiply the concentration by the dilution factor to get the actual concentration of your protein sample. Usually protein concentration is expressed as µg/mL or mg/mL so you may convert them in % if you consider 1mL is 1g (1 µg/g is 0.0001% and 1mg/g is 0.1%).
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