BIOL 1111
NB: Tables: 4-1; 4-2; 4-3 were empty and filled from the lab.
Data for Table 4-4 were obtained from the graph
UNIT 4: Quantitation of Protein Using Bradford Assay
Introduction
Many physiological and biochemical investigations require determination of the amount of total protein in a sample. It is impossible to place biological material under a microscope and count the number of protein molecules per unit volume the way we can count the number of cells or microorganisms. It is also impractical to weigh the solid protein after evaporating the solvent as the amount of protein we measure is often far below the detectable range by the most precise weighing instrument. Therefore, something measurable that is proportional to the concentration of the protein must be identified.
The measurement most commonly used in protein assays is the absorbance of light based upon Beer’s law, which states that when a solute absorbs light of a particular wavelength, the absorbance is directly proportional to the concentration of the solute in solution. However, proteins do not absorb sufficient light to assay by themselves. To make protein molecules absorb sufficiently measurable amount of light, a color reagent (dye) is chemically bound to the protein molecule and the absorbance of light by the dye molecule is measured.
In this lab, we will use a colorimetric spectrophotometry to determine protein concentration. In this technique, a dye reagent that binds to protein is added to protein sample and the amount of light absorbed by the dye-protein complex is measured by an instrument called spectrophotometer. Absorbance values from a set of protein samples with known concentrations are plotted to create a standard curve on linear graph paper. Protein concentrations of test samples can then be interpolated on a standard curve by hand or using a graphing utility such as Microsoft Excel.
Colorimetric Assay
A colorimetric assay refers to a method of determining the concentration of a chemical element or compound in a solution using a color reagent. There are several colorimetric methods for determining the total protein content of a sample: biuret, Lowry and Bradford. The biuret is the oldest method and is commonly used in biology teaching labs to detect the presence of a protein. It is the least sensitive of the three methods. The Lowry method is more sensitive than the biuret assay; however, the Lowry assay is affected by interference from many common laboratory reagents and chemicals. The Bradford protein assay is the most sensitive of the three and used most commonly. The Bradford assay uses a dye, Coomassie Brilliant Blue G250, which was first described by M. Bradford in 1976.
The Bradford Assay
The Bradford assay 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. The dye binds to proteins using three types of interactions. The primary interaction of the dye with proteins occurs through arginine, a very basic amino acid. Other weaker dye-protein interactions include the interaction of the aromatic rings of Coomassie Brilliant Blue G-250 dye with the aromatic rings of amino acids, such as tryptophan. Finally, the dye also weakly interacts with polar amino acids that have hydrophobic side chain such as tyrosine.
Coomassie Brilliant Blue G-250 exists in multiple forms. The dye in acidic condition takes on a reddishbrown color (cationic form) and 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 blue form (anionic form), and the absorption maximum shifts from 470 nm to 595 nm.
There is a correlation between the amount of blue color and the amount of protein in the sample and the intensity of the blue indicates the level of protein in a sample. The more intense the blue, the more protein present in the sample. The simplicity of the assay allows the results to be measured qualitatively by eye, or quantitatively with a spectrophotometer. By using a dilution series of proteins with known concentration, one can generate a spectrophotometric standard curve. The curve can then be used to estimate the quantity of protein in an unknown sample, based upon the intensity of blue.
The Bradford protein assay is simple, highly sensitive, and relatively less susceptible than other method to interference by many common laboratory reagents and chemicals. However, sodium dodecyl sulfate (SDS), a common detergent, which is widely used to lyse (break down) cells by disrupting the membrane lipid bilayer during protein extraction, interferes with the assay. Therefore, no SDS should be used in preparation of protein sample to be assayed by Bradford method. Another disadvantage of the Bradford assay is that it has a short linear range; i.e. the increase of the absorbance due to the increase in the protein concentration is linear only in a low concentration range, typically from 0 to 2 mg/ml. Therefore, dilutions of sample are often necessary before assay.
The Bradford assay is easy to perform and involves four main steps:
1. Preparation of a dilution series of known protein standards and preparation of unknowns.
2. Addition of reddish-brown Bradford dye (cationic form) and incubation for >5 minutes (but, not to exceed 60 minutes) for binding reaction of dye to protein, resulting in color change to the blue dye form (anionic form)
3. Quantitative reading of the absorbance at 595 nm in a spectrophotometer
4. Compilation of the data into a standard curve and determination of unknown protein concentration.
Standard Curve
A standard curve of a substance is constructed by measuring the absorbance of a series of different known concentrations of the substance and graphing the results by plotting absorbance on the Y-axis and concentration on the X-axis (See Figure 4-1). The unknown concentration can be determined from the standard curve by drawing a horizontal line on the graph parallel to the X-axis and through the point on the Yaxis that corresponds to the absorbance. This line will intersect the standard curve; at this intersection, a vertical line is dropped to the X-axis and the concentration read from the X-axis.
Two factors are important in determining concentrations using spectrophotometry. The absorption
maximum (a certain wavelength at which a substance absorbs the maximum amount of light) should be used, and absorbance rather than percent transmittance should be plotted because while absorbance is linearly proportional to concentration, transmittance decreases exponentially as protein concentration increases.
Absolute Concentration And Relative Concentration
In spectrophotometry, absorbance readings can be used to estimate the absolute or relative concentration of a substance in solution. To estimate the absolute concentration of a substance, the same substance of a known concentration should be available. To estimate absolute concentration of a substance with unknown concentration, a standard curve of known concentrations of the same substance should be constructed, and then the concentration of the substance of interest is interpolated from the curve using the absorbance reading of the substance with unknown concentration. In the previous lab period, you determined the absolute concentration of a methylene blue solution by constructing a standard curve using methylene blue solution with a series of known concentrations.
On the other hand, when the substance of known concentration is not available, or the substance of to-be measured is a mixture of different substances, a similar substance with known concentration is used for construction of a standard curve. Then the absorbance reading of the substance with unknown concentration is measured to interpolate the relative concentration of the substance from the standard curve.
FIGURE 4-1. Example standard curve having ten data points (known concentrations). The line is best-fit for the entire set of standard data points. Dashed arrow lines represent interpolations for a test sample having absorbance 0.6, which correspond to 560 µg/ml of -globulin on the X-axis.
Q 4-1. Is the concentration of -globulin determined on a BSA standard curve absolute or relative?
Exercise 1. Determination of Protein Concentration
In this laboratory exercise, the Bradford assay is used to determine concentrations of two protein samples using a gamma globulin (-globulin) protein standards.
You will:
1. construct a protein concentration standard curve using -globulin.
2. measure absorbance of protein samples: -globulin and BSA (bovine serum albumin) sample with unknown concentration, respectively.
3. determine the concentration of each protein sample by interpolating on the -globulin standard curve.
4. compare the results and discuss about the differences.
NOTE: The -globulin standard curve are linear typically at the concentration range between 0.2 – 1.5 mg/ml; therefore, the protein sample needs be diluted if the measured concentration falls beyond the upper linear range.
Activity A: Constructing -globulin Standard Curve
CAUTION! The dye reagent contains 10% phosphoric acid and 5% methanol. Avoid contact with skin or eyes. In case of contact, flush affected area with a plenty of water.
1. Turn on the spectrophotometer and set wavelength at 595 nm.
2. While the spectrophotometer warms up, obtain the following items:
2a. Eight (8) clean 1.5 ml microcentrifuge tubes on a microcentrifuge tube rack.
2b. Eight (8) disposable cuvettes on a cuvette rack & eight (8) reusable cuvette caps.
2c. Obtain a 1.5 ml microcentrifuge tube containing 400 µl of 2.0 mg/ml -globulin protein standard stock solution. Keep the tube on ice using a floating rack provided on your lab bench.
2d. Obtain a 1.5 ml microcentrifuge tube containing 1 ml of phosphate buffered saline (PBS) solution. Keep the tube on ice along with standard stock solution on the floating rack.
2e. Obtain a cap-less 20-ml vial and dispense 15 milliliters of Bradford protein dye reagent from the dispenser bottle on the supply table and keep the vial at room temperature.
NOTE: Your results will only be as good as your micropipetting technique and consistency in handling samples! Be careful not to cross-contaminate the concentration standards.
Accurate measurement of standard solution is very important to construct a good standard curve. In consideration of the viscosity of protein solutions, aspirate and distribute the sample carefully and slowly. Remember one-second-pause rule for aspiration and distribution.
Activity A: Constructing -globulin Standard Curve
To dispense the last drop of sample clinging at the very end of the tip, touch the dry spot of the inside wall of the tube with the end of the tip while distributing and purging the sample, and slowly remove the tip without drawing the liquid on the tube back into the pipette tip.
If you fail to obtain a straight standard curve, you must repeat the procedure until you get one approved by your lab instructor.
3. Prepare 100 µl of protein standard solution in 8 different concentrations, including a ‘blank’ as follows:
NOTE: The standard solution containing no protein (0 mg/ml; tube ‘0’) is called a ‘blank.’ The blank contains only the PBS solution, which is used to prepare the protein standard solution and protein samples.
3a. Fill in the blanks in Table 4-1 to determine the amount of protein standard stock solution and diluent (PBS solution) to be added in each concentration standard.
TABLE 4-1. Dilution of standard stock solution of -globulin to make 100 µl protein standard solutions.
Stock Conc. = 2.0 mg/ml Final Volume = 100 µl per sample
Tube No. Final Conc. (mg/ml) Vol. of Stock to add (µl) Vol. of PBS to add (µl)
0a 0.0 0 100
1 0.2 10 90
2 0.4 20 80
3 0.6 30 70
4 0.8 40 60
5 1.0 50 50
6 1.2 60 40
7 1.4 70 30
a. blank
3b. Label eight (8) 1.5 ml microcentrifuge tubes ‘0’, ‘1’, ‘2’, ‘3’, ‘4’, ‘5’, ‘6’, and ‘7’ to indicate the following concentrations of protein standard: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg/ml, respectively (See Table 4-1).
3c. Pipette appropriate amount of the standard stock solution into each tube.
NOTE: You may keep using the same pipette tip for all concentration standards as long as you add the lowest concentration first followed by the next higher concentration. However, if a sample begins to adhere to the wall inside a pipette tip, you should use fresh tip to deliver accurate amount of the solution.
3d. Using a fresh tip, add appropriate amount of PBS solution to each tube. While you adding PBS
solution, be careful not to touch the spot in the tube you previously touched to add standard solution.
3e. Cap the tubes tightly and mix thoroughly by one of the following methods:
1) repeatedly flickering the tube with your finger.
2) briefly (2-3 seconds) shaking on a vortex mixer.
3f. Place the tubes containing protein standard solutions on the ice.
4. Carry out color reaction for protein standard solutions as follows:
NOTE: Do not shake a cuvette containing sample solution. Foams and bubbles will interfere with the light passage through the cuvette and cause unstable and erratic readings.
Since the color reaction starts immediately when standard solution (containing protein) and the color reagent make contact each other, it is important to add and quickly mix each reaction without a delay.
As the color reaction continues after the incubation period, and will increase absorbance, samples should be read at the same time interval without delay between samples.
Try a ‘dry-run’ the entire procedure to familiarize yourself to the entire procedure before running your samples as the reaction and measurement must be run without an interruption.
4a. Label eight (8) disposable cuvettes to indicate the concentrations of protein standards as in Step 3b above.
4b. Place the labeled cuvettes on a cuvette rack. Do not lay them down on the lab bench.
4c. First, add 1.0 ml of the dye reagent to each cuvette from the vial.
4d. Accurately pipette 20 µl of the appropriate standard into each cuvette one by one at one-minute interval. Record the time upon each standard was added to follow exact incubation time (Table 4-2).
4e. Immediately and quickly cover the cuvette with a cuvette cap and mix thoroughly by gently (but quickly) inverting the cuvette a few (2-3) times.
4f. Place the cuvette back on the cuvette rack and incubate it at room temperature so that dye-protein binding reaction takes place in each sample exactly for 10 minutes from the moment the standard protein solution was added to the dye. If the time interval is not exactly one minute, carefully track time so that you incubate each reaction for the same period of time.
NOTE: If you incubate the standards longer than one minute, you should do so with your protein samples.
5. As soon as the incubation time for the blank is 10 minutes, measure absorbance of the each standard at 1minute interval (or at the same interval you added standard into the cuvette) sample as follows:
5a. Insert the cuvette containing blank solution, and press ‘Blank’ button to calibrate the spectrophotometer.
5b. Record absorbance reading for the blank, which should read ‘0.000’. The third digit following decimal point might fluctuate by a few units; but it is normal. However, if the reading is 0.010 or higher, re-blank the spectrophotometer repeatedly until you obtain a reading close to 0.000.
CAUTION! Do not discard blank after the initial calibration of spectrophotometer since it will be used again to recalibrate the spectrophotometer before measuring absorbance of protein samples.
Activity A: Constructing -globulin Standard Curve
NOTE: Absorbance reading must be taken at the same interval as the reaction interval used above, which was set to 1 minute for convenience.
5c. Measure and record absorbance of each standard in Table 4-2 at the exactly same time interval.
TABLE 4-2. Time of sample addition and absorbance reading, and absorbance readings of -globulin-Bradford dye complex at 595 nm.
Cuvette No. -globulin Conc.
(mg/ml) Time of Sample Addition (min.) Time of Abs. Reading (min.) A595
0a 0.0 0 10 0.00
1 0.2 1 11 0.114
2 0.4 2 12 0.193
3 0.6 3 13 0.261
4 0.8 4 14 0.379
5 1.0 5 15 0.515
6 1.2 6 16 0.621
7 1.4 7 17 0.714
a. blank
5d. Plot a graph (standard curve) of the absorbance at 595 nm (A595) on Y-axis versus the concentration of the protein standard on X-axis. See information and example in the introduction section of this exercise.
5e. Show your standard curve to your lab instructor. If the standard curve is acceptable (i.e., reasonably straight in slope), proceed to ““Activity B:” on page 9” to determine concentrations of protein samples.
NOTE: If you need to repeat standard curve construction, discard all used samples including the blank, obtain new standard stock and Bradford reagent, and do it all over again from scratch.
Activity B: Determining Protein Concentrations
Activity B: Determining Protein Concentrations
NOTE: To avoid cross-contamination between your samples, you must use a fresh pipette tip and a fresh tube for each sample.
1. Obtain two (2) clean 0.5 ml microcentrifuge tubes and label them ‘G’ and ‘A’ to represent -globulin and bovine serum albumin, respectively.
2. Pipette 100 µl each of protein samples in the appropriately labeled tubes at the supply station. Returning to your lab bench, always keep the protein samples on ice.
3. Obtain six (6) disposable cuvettes, and label them G1, G2 and G3 to indicate triplicates of g-globulin samples, and A1, A2, and A3 for bovine serum albumin sample triplicates.
4. Carry out color reactions by mixing 1.0 ml dye reagent and 20 µl protein sample in the cuvette, followed by 10-minute incubation.
NOTE: As the intensity of the color depends on time and the color reaction continues after the dye reagent and protein sample are mixed, you should add and mix all reactions and measure absorbance at the same time interval as you did for the standards.
5. Blank the spectrophotometer using the same blank cuvette used for construction of the standard curve.
6. Measure A595 for each sample filling spaces in Table 4-3.
7. Determine the concentration of each protein sample and fill the space in Table 4-4 using the hand-drawn standard curve constructed above.
8. Concentrations of the triplicates of a protein sample should be close to each other. If they are different each other by more than 0.1 mg/ml, you must re-do the assay. Duplicate Table 4-3 and Table 4-4 in your lab notebook as necessary to record the data for repeated assay(s).
Q 4-2. Samples of -globulin and BSA used in this experiment were prepared at the same concentration. Were the concentrations of your -globulin and BSA samples determined by Bradford assay the same or close to each other? If different, how much and why?
Consider the binding characteristics of Bradford assay reagent (Coomassie Brilliant Blue G-250 dye) to different amino acids in the protein and the amino acid composition in -globulin and BSA. You must search the literature to find scientifically feasible explanation beyond what is described in this manual.
The Introduction section of your lab report must include statements about the binding characteristics of Bradford assay reagent and the amino acid composition in -globulin and BSA, as well as your prediction. The Result section of your lab report must include a sentence(s) stating whether the two protein concentrations were close to each other or how significantly different. And, you must provide the reasons to have the results in the Discussion section in your report. Your explanation should also be summarized in a sentence in the Abstract of your report.
Activity B: Determining Protein Concentrations
TABLE 4-3. Absorbance readings of -globulin and bovine serum albumin at 595 nm using Bradford assay.
Cuvette
ID. Time of Sample Addition (min.) Time of Abs. Reading (min.) A595
0a 09:00 0.000
G1 0:00 10:00 0.587
G2 01:00 11:00 0.447
G3 02:00 12:00 0.509
A1 03:00 13:00 0.864
A2 04:00 14:00 0.868
A3 05:00 15:00 0.860
a. same blank used for standard curve.
TABLE 4-4. Concentrations of -globulin and bovine serum albumin
determined by a hand drawn -globulin standard curve using Bradford assay.
Protein Sample Protein
Conc. (mg/ ml)a Average
G1 1.11 1
G2 1.01
G3 0.89
A1 1.70 1.7
A2 1.70
A3 1.70
a. Protein concentrations are Interpolated from the -globulin standard curve.
Activity C: Using Excel Program To Estimate Protein Concentration
Using Excel To Construct A Standard Curve
In a conventional standard curve for determination of protein concentration, the absorbance values are plotted as the dependent variable on the Y-axis, and the concentrations as the independent variable on the X-axis (See Figure 4-1 on page 3) with relationship equated as follows:
2
y = ax + +bx c [EQ 1]
where, we need to solve for x to determine the protein concentration of the sample.
But, if we switch the axes and plot absorbance on the X-axis and protein concentration on the Y-axis, the protein concentration is represented by y and the equation is much easier to solve.
To use Excel program (Office 2010 version) for generating such an equation:
1. Enter the absorbance data for the standard curve in a column and concentration data in the column next to it (Column A and B in the figure below), including only those fall within a linear range of the standard curve.
2. Highlight both columns, including concentrations of standards you used for drawing a best-fit line and corresponding absorbance readings.
Activity C: Using Excel Program To Estimate Protein Concentration
3. From [Insert] menu tab, click the inverted triangle to right of the scatter plot icon and select
Alternatively, from [Insert] menu tab, click the diagonal arrow at the bottom right corner of the chart menu group to open ‘Insert Chart’ dialog box and then select
4. To obtain a trendline:
4a. Select the resulting graph (click on the edge of the chart, if not selected yet).
4b. Under the [Chart Tools], select [Layout] tab.
4c. Click [Trendline] and then select
4d. Under the [Trendline Options] tab, select “Linear”, and check “Display Equation on chart”.
5. Now, you may use the resulting equation to determine protein concentration (y) of an unknown sample by inserting the sample’s absorbance value (x).
Activity C: Using Excel Program To Estimate Protein Concentration
Using Excel To Determine Protein Sample Concentrations
Using the equation generated from the standard curve in Excel, concentrations of protein samples can be readily determined without interpolating on the hand-drawn standard curve.
In Excel:
1. Enter sample ID’s in a separate column (e.g., column E)
2. Enter absorbance of each sample in a separate column (e.g. Column F) under a heading “Absorbance” or “A595”.
3. Right to the Absorbance column, prepare a column with a heading for concentration, such as “Conc. (mg/ ml)”.
4. Enter the formula (right side of the equation containing x variable) in Column G under the heading “Conc
(mg/ml)” replacing the x variable with spreadsheet cell id from the column of ‘A595’.
Formula displayed on the chart:
0.7273x – 2E-16
Format of formula to be entered in the spreadsheet cell:
0.7273*F3 – 2E-16 or
0.7273*F3 – 0.0000000000000002
where, ‘F3’ is the id of the cell containing absorbance reading of the first fraction in the column of ‘A595.’
5. Copy the cell in which you just entered the formula, and paste it in the rest of the cells in the column to match the absorbance values on the left side.
