Why is it necessary to construct a protein standard curve in the Lowry assay?
Constructing a protein standard curve is of prime importance in studying the activity of an enzyme as this analysis relies on accurate quantitation of protein concentration.
How do you use a standard curve to determine protein concentration?
Plotting a graph with the absorbance value as the dependent variable (Y-axis) and concentration as the independent variable (X-axis), results in an equation formatted as follows: y = ax2 + bx + c, where solving for x determines the protein concentration of the sample.
Is a standard curve required for Bradford assay?
The traditional method for calculating protein concentration of an unknown sample is to use a standard curve that is generated from known protein standards.
How do you find the standard curve?
Standard Curves To calculate the sample concentration based on the standard curve, first you find the concentration for each sample absorbance on the standard curve; then you multiply the concentration by the dilution factor for each sample.
Why BSA is used as a standard?
BSA is used because of its ability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
Why BSA is used in Lowry method?
Here, it was aimed to estimate the protein content by Lowry’s method at different wavelengths by UV-visible spectrophotometric analysis. Using Bovine Serum Albumin (BSA) particularly as a standard protein estimation of proteins is done because of its low price, high purity and ready availability.
What is the basic principle of Lowry method?
Principle: The principle involved in Lowry method is determining the protein concentration by calculating the reactivity of the peptide nitrogen with the Copper ions under alkaline conditions followed by reduction reaction of Folinciocalteay phosphomolybdic phophotungstic acid to Heteropolymolybdenum blue by copper …
How do you calculate protein concentration using the Lowry method?
Add 4.5 ml of Reagent I and incubate for 10 minutes. After incubation add 0.5 ml of reagent II and incubate for 30 minutes • Measure the absorbance at 660 nm and plot the standard graph . Estimate the amount of protein present in the given sample from the standard graph.
How do you find the standard curve of a concentration?
The equation y=mx+b can be translated here as “absorbance equals slope times concentration plus the y-intercept absorbance value.” The slope and the y-intercept are provided to you when the computer fits a line to your standard curve data. The absorbance (or y) is what you measure from your unknown.
What is a standard curve graph?
Standard curves are graphs of light absorbance versus solution concentration which can be used to figure out the solute concentration in unknown samples. We generated a standard curve for a set of albumin samples. Interpreting a Standard Curve. A spectrophotometer measures light quantity.
What is a reasonable range of BSA concentrations for a Bradford assay standard curve?
0 – 1 mg/ml
For BSA, use 0 – 1 mg/ml as your standard curve concentration; for IgG, use 0 – 1.6 mg/mL.