The Bradford protein assay begins with the addition of A BSA standard in increasing quantities and dilutions of an unknown sample to a 96 well plate deionized water is added to complement the variable volume. The Bradford reagent is then added to all wells except for the blank, which must be water after five minutes. Absorbance is read at 590 nanometers for the detection of protein dye complex and at 450 nanometers.
For linearization of the graph, the ratio of absorbance values at 590 nanometers and at 450 nanometers is strictly linear with protein quantity. Hi, I'm from the Department of Biochemistry at Tel Aviv, university of Israel, I'm or T from the lab. Today we will show you a procedure for determining protein concentration using an improved Linearized Bradford protocol.
We use this procedure in our laboratory to normalize protein quantity in SZaS, for example, before we load gels. So let's get started. In the Bradford Assay, three forms of the Kumasi brilliant blue dye are an acid-based equilibrium at the usual acidic pH of the assay.
The red, blue and green forms have absorbent maxima at four hundred and seventy five hundred ninety and 650 nanometers respectively. The blue is the form that binds the protein forming a complex that intensely absorbs light at 594 nanometers. This widely used assay has a large degree of curvature over a broad range of protein concentrations.
Therefore, only a narrow range of relatively high protein concentrations is used for the calibration graph, which then better fits linear regression. One source for the non-linearity is the absorbence at 590 nanometers of the free dye, which decreases as increasing protein quantities are added. Non-linearity is partially reduced by measuring the change of absorbence at 450 nanometers where the diet itself, but not the protein dye complex absorbs.
And by reducing the correct calculated blank, the decrease in free dye concentration produces another distortion of the linear response because as protein dye binding is in equilibrium, complex formation depends not only on the concentration of the free protein, but also on that of the free dye. Taking into account both issues related to the variable concentration of the free dye, a mathematical equation was developed that describes a linear relationship between protein concentration and the ratio of absorbance measurements. 590 nanometers over 450 nanometers.
The mathematical equation was experimentally tested and found to yield a linear calibration curve over the entire protein concentration range. To begin, prepare a 0.1 milligram per milliliter stalk solution of the standard bovine serum albumin. Any other standard may be chosen, but note that the same standard must be used in all experiments.
Dilute the unknown samples in deionized water. Aim for five to 50 micrograms per milliliter, yet higher or lower protein concentrations are acceptable since there is no apparent limit for the linear range of the assay. Note however, that the measurement must be within the linear range of the absorbance reader.
Also dilute the Bradford reagent 2.5 fold in deionized water. Next, add zero and five to 50 microliters of the BSA stock solution to triplicate wells of a 96 well plate to create a zero to five microgram BSA calibration curve complement with deionized water to reach 100 microliters per well in different wells. At 100 microliters of unknown sample in triplicates, several concentrations of the unknown sample may be used to increase accuracy.
Finally, at 100 microliters of the diluted Bradford reagent to all wells. The total volume is 200 microliters per well. Also prepare a blank for measuring the absorbance.
This must be 200 microliters of deionized water and not the zero protein dye. Well wait at least five minutes, but not more than 60 minutes. For color development.
Proceed to measure the absorbance at 590 nanometers and at 450 nanometers. Remember to use water for the blank measurement. Prepare a calibration graph by dividing the net absorbance values at 590 nanometers and at 450 nanometers.
Note that the zero protein value should be included as a data point. Calculate the concentration of the unknown sample based on the linear equation of the calibration curve. Unlike the absorbance at a single wavelength of 590 nanometers, the ratio of absorbance values 590 nanometers over 450 nanometers is linear with protein concentration.
The protein concentration of the unknown sample may be simply calculated using the linear equation of the calibration curve. However, increased accuracy is obtained by measuring several unknown sample dilutions. To this end, prepare two graphs.
The first is a calibration graph for the standard with micrograms of protein on the x axis. The second graph is for the unknown sample with microliters of the original undiluted sample on the x axis. The dye only value should be included in both graphs.
The protein concentration of the unknown sample is derived by dividing the slopes of the unknown sample and the standard. We have just shown you how to measure protein concentration using an improved linearized Bradford protocol. Using this procedure greatly increases the accuracy of the assay, improves the sensitivity about 10 folds, and significantly reduces interference by detergents Doing this procedure.
It's important to remember to use the appropriate blank that is water rather than the zero protein dye sample. So that is it. Thank you for watching and good luck with your experiments.
