Lab 3: Bradford Assay

Biuret Method

- Oldest method and is commonly used in high school labs to detect the presence of a protein. - It involves two reactions; a chelation and a redox reaction. - It is the least sensitive of the three methods.

Excel Review

- Open Excel and give the sheet a title. In this case, "Got Protein?". - Set up columns for Tube #, Standard Concentration and Absorbance. - Tubes numbers should include a Blank and tubes 1 - 7. - Known standard concentrations should be placed into the appropriate column. Concentrations are in mg/ml. - Recorded absorbance values should be entered into the Abs. column. - Columns should also be set up to inter your unknown sample data. - Enter your unknown letter and the absorbance for each. Remember to add a column for the dilution factor of 50. your unknowns were diluted and you will need to multiply it by 50 to get the correct concentration. -

Beer's Law

- States that when a solute absorbs light of a particular wavelength, the absorbance is directly proportional to the concentration of the solute in solution. - The measurement most commonly used in protein assays is the absorbance of light. However, proteins do not absorb sufficient light to assay -— by themselves.

The Bradford Assay Limitations

- The assay measures total protein concentration, different methods must be used to identify specific proteins. - Assay is linear over a limited range. - The Coomassie dye binds specifically to arginine and hydrophobic amino acids. - The amino acid composition can alter the concentration-absorbance curve. Use of a standard (like BSA-Bovine Serum Albumin) with a similar composition must be used.

Bradford Protein Assay Method

- The most sensitive of the three. - Uses a dye, Coomassie Brilliant Blue G250, which was first described by M. Bradford in 1976. - Takes advantage of the chemical properties of the dye and the dyes ability to interact with the side chains, or R-groups, of specific amino acids.

Coomassie G-250 Binds to Proteins Using Three Types of Interactions

- The primary interaction occurs through arginine, a very basic amino acid; interacts with negatively charged sulfate groups through electrostatic interactions. - Weaker dye-protein interactions: Aromatic rings of Coomassie G-250 dye with the aromatic rings of amino acids, such as tryptophan, through electron stacking interactions. - Finally, the dye also weakly interacts with polar amino acids that have hydrophobic R-groups, such as the aromatic ring of tyrosine.

Beer's Law Formula

A = ε × b × c ε - the molar absorptivity (L mol-1 cm-1) b - the path length of the sample (usually 1cm-cuvette) c - the concentration of the compound in solution (mol L-1)

Which of the following colorimetric methods for determining the total protein concentration of a sample is the most sensitive? A) Bradford assay B) Lowry method C) Scrooge and Marley assay D) Biuret assay

A) Bradford assay

Coomassie G-250 dye binds to proteins using 3 types of interactions. The primary interaction of the dye with proteins occurs through ____________________. A) Glycine B) Arginine C) Leucine D) Lysine

B) Arginine

Beer's Law states: A) Dyes react with proteins B) The absorbance of light is directly proportional to its concentration C) Proteins absorb light at different wavelengths. D) Three types of beer is the maximum you should drink in one night

B) The absorbance of light is directly proportional to its concentration

Colorimetics assays use _______________________ created by measuring _______________________ of solutions of known concentrations to determine the concentrations of unknown samples. A) color; chemical reactions B) standard curves; absorbances C) emissions; absorbances D) concentrations; signal

B) standard curves; absorbances

Colorimetric Methods for Determining the Total Protein Content of a Sample

Biuret, Lowry and Bradford.

The Bradford assay will be used to collect absorbance data on the samples and standards. The correct volumes/ratio used to prepare the samples is: A) none of the above B) 1mL of sample or standard and 20uL of reagent C) 20uL of sample or standard and 1 mL of reagent D) 20uL of reagent and 1 mL of sample or standard

C) 20uL of sample or standard and 1 mL of reagent

In the Bradford assay, the peak absorbance of unprotonated Coomassie G-250 is at A) 475 nm B) 495 nm C) 620 nm D) 595 nm

D) 595 nm

After reviewing the following standard curve you notice that: A) The assay is set to Bradford assay, the cuvette size is set to 4mm, and each of the standards has been run 4 times B) The assay is set to Lowery assay, the cuvette size is set to 5mm, and each of the standards has been run 6 times C) The DeNovix is set to Bradford assay, the cuvette size is set to 4mm, and each of the standards has been run 5 times D) The assay is set to Bradford assay, the cuvette size is set to 5mm, and each of the standards has been run 5 times

D) The assay is set to Bradford assay, the cuvette size is set to 5mm, and each of the standards has been run 5 times

Biophotonics means: A) Taking pictures of biological materials. B) Biological molecules change color. C) Biology depends on light. D) Using the interaction of biological materials and light to study those materials.

D) Using the interaction of biological materials and light to study those materials.

The Bradford's Assay is a colorimetric assay, meaning there is a color change in the dye. What are the colors? A) from clear to brown B) from blue to brown C) from clear to blue D) from brown to blue

D) from brown to blue

If you need to dilute your samples, what should you dilute them with? A) 10% Tris buffer B) Bradford's dye C) 1x PBS D) water

D) water

Protein Quantitation

Often necessary before isolation, separation, and analysis by chromatography, electrophoresis, or western blotting.

Ways Light can Interact with Biomolecules

Reflection, absorption, transmission, and light scattering.

Biophotonics

The technology that focuses on the interaction of biological materials with light and other forms of radiant energy whose quantum unit is the photon.

T or F: The Blank will also be used as the 0 ug/ul reading for the standard curve.

True

T or F: The Lowry method involves two redox reactions and is more sensitive that the Biuret assay.

True

T or F: You will need to re-blank the DeNovix following each of the samples or standards during data collection

True

Formula for diluted concentration of the sample?

x=((y - 0.0586)/0.541) , then multiply it by *50 to get the actual concentration of your unknown. - Just remember to put the cell number of your abs. data in place of the Y.

Data Analysis

ü The following graph illustrates a representative standard curve that can be generated in this exercise. ü This graph shows the raw absorbance data was plotted (absorbance vs. concentration), and a best-fit curve was. ü The high R2 value depicted for this curve (R2 = 0.98) illustrates the stronglinearity of these data. ü Correlation coefficients of x >0.95 reflect data which exhibit a high degree of linearity and can be used to accurately estimate unknown values. üTo generate the standard curve, the measured absorbance of each standard in the curve is plotted against the known protein concentration. ü The resulting standard curve can be used to estimate the concentration of an unknown protein based upon its measured absorbance value. ü Alternatively, if a spectrophotometer is not available, you could compare your unknown samples qualitatively to a dilution series to determine the protein concentrations.

The Bradford Method

• Is based on the color development formed when the dye Coomassie Blue G-250 binds to protein. • The unique chemical properties of the dye allow it to interact with the side chains, or R-groups, of specific amino acids. • There is a correlation between the amount of blue color and the amount of protein in the sample: the more protein, the more intense the blue color. • The simplicity of the assay allows the results to be measured qualitatively by eye, or quantitatively with a spectrophotometer. • In this lab, you will use absorbance values from a set of protein samples with known concentrations to create a standard curve on linear graph paper. • Protein concentrations of your test samples can then be extrapolated by hand or plotted using a graphing utility such as Microsoft Excel.

Peak absorbance of Unprotonated Coomassie G-250 dye?

595 nm

Coomassie G-250 in B.A.M.

- Exists in its cationic state and takes on a reddish-brown color. The peak absorption of the dye in this state is 470 nm. - When the dye binds to and interacts with amino acids, the dye is converted to a stable unprotonated blue form, and the absorption maximum shifts from 470 nm to 595 nm. - This stable blue form of the dye is easily observed and quantified in a spectrophotometer.

Creating a Graph for Standard Curve

- In order to create a graph of for your standard curve you will need to highlight both the concentration and absorbance columns. - Then go to the top tabs and click insert. - Then click on Scatter for your scatter plot. - Click on scatter with only markers. - You should get a graph that looks like the one in the screenshot.

Lowry Method

- Involves two redox reactions. - It is more sensitive than the biuret assay; however, the Lowry assay is affected by interference from many common laboratory reagents and chemicals.

4 Main Steps of B.A.

• Preparation of a dilution series or serial dilutions of known protein standards and preparation of unknowns. • Addition of Bradford dye (brown, cationic form) and incubation for >5 minutes (not to exceed 60 minutes). • Binding of dye to protein, resulting in color change to the blue, unprotonated dye form and quantitative reading of the absorption at A595 in a spectrophotometer. • Compilation of the data into a standard curve and unknown protein concentration determinatio