Explain the basis of Lowry method briefly ?
The Lowry method is a common biochemical assay used to measure the concentration of proteins in a solution. It is based on the reaction of proteins with copper ions (Cu2+) in an alkaline solution. The reaction between the peptide bonds in proteins and the Cu2+ ions results in the formation of a purple-colored complex.
Here is a brief explanation of the Lowry method:
1. Sample Preparation: Start by preparing your protein sample. It can be extracted from cells, tissues, or any other biological material. Make sure to homogenize and lyse the sample to release the proteins.
2. Protein Reaction: In a test tube, add your protein sample to a mixture of Folin-Ciocalteu reagent and sodium carbonate solution. The Folin-Ciocalteu reagent contains phosphomolybdate and phosphotungstate, which are reduced by the Cu2+ ions derived from the protein reaction. This reduction leads to the formation of a blue chromophore.
3. Incubation: After adding the reagent, let the reaction mixture incubate at room temperature for a specific period of time. During this time, the Cu2+ ions react with the proteins in the sample.
4. Measurement: After the incubation, measure the absorbance of the blue chromophore at a specific wavelength (e.g., 750 nm) using a spectrophotometer. The absorbance is directly proportional to the concentration of proteins in the sample.
5. Standard Curve: To determine the protein concentration in your sample, prepare a series of standards with known protein concentrations. Measure their absorbance using the same wavelength as the sample. Plot a standard curve using the absorbance values of the standards and their corresponding protein concentrations.
6. Calculation: Finally, use the standard curve to determine the protein concentration in your sample by comparing its absorbance to the absorbance values of the standards. Interpolate the unknown protein concentration from the standard curve.
Remember, protein assays such as the Lowry method should be optimized based on the type of protein and the desired sensitivity of the assay. Adjustments to reagent concentrations and reaction time may be necessary to obtain accurate and reliable results.