General Biochemistry Laboratory Protein Quantitation Assays Objectives Upon completion of this experiment, students should be able to: • describe how colorimetric and absorbance assays can be used to determine protein concentration.

describe how the biuret and Bradford assays work. generate a standard curve by diluting a protein stock solution use biuret and Bradford assays together with a standard curve to determine the concentration of a protein sample. I. Background: Estimating the concentration of a protein solution using its extinction coefficient based on Beer's Law is a practical method that is commonly used in biochemistry laboratories (See Experiment #1). But if you do not know the extinction coefficient of the protein, how can you estimate its concentration? This experiment explores other alternatives to the Beer's law approach. It is frequently necessary to measure protein concentrations in biochemistry: to determine the specific activity of an enzyme; the stoichiometries of proteins and the molecules they interact with; or to measure the purity of a particular protein from a mixture as a percent of the total. The most accurate method for measuring a purified protein is by quantitative amino acid analysis of a hydrolyzed protein in conjunction with its known amino acid composition However, this is tedious, difficult, and not practical for everyday use. Fortunately, numerous colorimetric protein assays as well as various spectroscopic methods have been developed for routine estimation of protein concentration. The criteria for choice of a protein assay are usually based on convenience, availability of protein for assay, presence or absence of interfering agents, and need for accuracy. For example, the Lowry method is very sensitive but is a two-step procedure that requires a minimum of 40 minutes incubation time. The Bradford assay is sensitive and can be read within 5 minutes, however proteins with low arginine content will be underestimated. Generally, estimates are more accurate for complex mixtures of proteins. Estimates of concentration of pure proteins can be very inaccurate depending on the principle of the assay, unless the same pure protein is used as a standard. Because different proteins have different amino acid compositions, the sensitivity of colorimetric assays to individual proteins may vary widely. The most reproducible results are obtained with standards composed of a mixture of proteins that is as similar as possible to the unknown. A relative measurement is good enough for most purposes, but the standard used should be reported. For example, the Bradford assay is much more sensitive to bovine serum albumin (BSA) than to immunoglobulin G (IgG), so that with IgG the investigator is likely to underestimate the amount of protein in a sample, while with BSA the investigator is likely to overestimate the amount. Methods to estimate protein concentrations are based either on absorbance or colorimetry A. Absorbance Assay: The experiment that you carried out last week is the basis for these absorbance-based assays. These assays are dependent on the intrinsic absorbance of the protein. The intrinsic absorbance of a protein in the near-UV portion of the spectra is primarily dependent on the absorbance of the three aromatic amino acids (TIR, Tyr, and Phe). The absorbance of a solution is described by Beer's law: A = Elç REV_0818_RB/nwhere A is the absorbance at a particular wavelength, &, is the proportionality constant called the absorptivity or extinction coefficient of the species at the wavelength, I the path length light passes through the sample (typically 1 cm), and c the concentration of the absorbent (typically in mg/ml or M for molarity, depending on the absorptivity coefficient used). Below are a few of the absorbance-based assays: Absorbance at 280 nm . . Range of sensitivity: 20 micrograms to 3 mg Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater • Accuracy: Fair Convenience: Excellent, if equipment available Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets Absorbance at 205 nm Range: Roughly 1 to 100 micrograms Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater Fair Convenience: Very good Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets Extinction Coefficient Range: 20 micrograms to 3 mg Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater • Accuracy: ~2% (very good) Convenience: Very good Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets B. Colorimetric assays: The colorimetric assays are dependent on the interactions of proteins with organic or inorganic molecules that result in a change in the absorbance properties of the sample upon binding. For these assays standard curves are generated using a reference protein (eg., serum albumin). These curves are more or less linear with protein concentration. Comparison of the absorbance of the unknown protein sample with the equation of the linear regression line (which describes the standard curve) allows for calculation of the concentration of the unknown. Accuracy: Many types of assays have been developed, each with their own advantages and disadvantages. In selection of the most appropriate protein assay, several factors must be considered: ease of assay, sensitivity, accuracy, and the effect of interfering substances on the assay. Some characteristics of the most commonly used protein assays are listed below. Modified Lowry Range: 2 to 100 micrograms Volume: 1 ml (scale up for larger cuvettes) Accuracy: Good Convenience: Fair Major interfering agents: Strong acids, ammonium sulfate REV_0818_RB/nBiuret Bradford as say Range: 1 to 20 micrograms (micro assay); 20 to 200 micrograms (macro assay) Volume: 1 ml (micro); 5.5 ml (macro) • Accuracy: Good . Range: 1 to 10 mg Volume: 5 ml (scale down for smaller cuvettes) Accuracy: Good Convenience: Good Major interfering agents: Ammonium salts Convenience: Excellent Major interfering agents: None Bicinchoninic Acid (Smith) . Range: 0.2 to 50 micrograms Volume: 1 ml (scale up for larger cuvettes) • Accuracy: Good • • Major interfering agents: Strong acids, ammonium sulfate, lipids Convenience: Good Amido Black method Range: 2 to 24 micrograms Volume: 2 ml Accuracy: Good Convenience: Poor • Major interfering agents: None reported Colloidal Gold Range: 20 to 640 nanograms (high sensitivity) Volume: 1 ml (scale up for larger cuvettes) Accuracy: Fair Convenience: Poor • Major interfering agents: Strong bases For today's lab, we will be determining protein concentration using two colorimetric protein assays: Bradford (or BioRad assay) and Biwet assay. Both assays will be done with a lab partner using visible-range spectrophotometers. JJA. BIURET PROTEIN ASSAY Background: This assay is named for biuret, a small compound that is formed by the condensation of two urea molecules upon heating. The amide groups formed in the condensation reaction bind to copper ions at basic pH. REV_0818_RB/nThe copper complexes that result from this interaction produce a strong blue color that can be measured with a spectrophotometer. Proteins also contain amide groups formed when an amino group and a carboxyl group join to form a peptide bond. Therefore, proteins will also complex with copper ions at a basic pH. Because this reaction was first observed with biuret, it is called the biuret reaction, and when this reaction is used to measure protein concentrations, it is called the Biuret Protein Assay. Thus, in this assay you will combine protein samples with biuret reagent which contains copper ions in a basic solution. The copper ions will complex with the amide groups in the proteins to create a blue color that will be measured using a spectrophotometer set to 550 mm. The amount of blue color that forms is directly proportional to the quantity of protein in your samples. The assay is not as sensitive as the Bradford assay so the standard curve will be generated with higher concentrations of standard protein. 0=¢ H-NI fogt O=C H-NI C-R R R- IN-H C=0 IN-HI Figure Cu* interactiwith peptide bonds Biuret Protein Assay Procedure: The objective of the assay is to collect data and plot a standard curve of absorbance vs. known concentration of proteins. Then you will be given a protein solution of unknown concentration and your goal is to determine its concentration using the standard curve. A. Information Necessary for Data Collection: a. A spectrophotometer will be set up for you in the lab b. You will collect data at 550 nm. B. Reagents: a. The biuret reagent will be prepared for you according the following procedure: A formula for biuret reagent is (per liter final volume) 9 gm Sodium potassium tartrate (fw. 282.22), 3 gm Copper sulfate x 5 H2O (fw. 249.68), 5 gm Potassium iodide (166.0), all dissolved in sequential order in 400 ml 0.2 M NaQH (mw 40.0) before bringing to final volume. The volume can be scaled up or scaled down. Discard if a black precipitate forms. (This has been prepared for you). b. A freshly prepared 20 mg/ml stock solution of bovine serum albumin protein will be provided. You will use this solution to prepare different dilutions of known concentrations for your standard curve. To determine the dilutions, you will use the dilution equation (M1V1 = M2V2). c. A protein solution of unknown concentration. c. Procedure: put your pipetting skills into practice! 1. Use the dilution equation to calculate the following dilutions: In clean test tubes prepare 1 mL each of six separate standard solutions from the stock solution of bovine serum albumin: 0 mg/ml (this is your blank, no protein) 1 mg/ml, 2 mg/ml, 5 mg/ml 7.5 mg/ml, REV_0818_RB/nand 10 mg/ml. Use the same buffer (0.1 M NaCl) in which the stock protein solution is dissolved. Label the tubes appropriately. 2. Separately, transfer 1.0 mL of your unknown solution to a clean, labeled test tube. It does not need to be diluted. We will use the standard curve generated from the six samples prepared above to determine the unknown concentration of this one. 3. Using the P1000 pipette, add 4 mL of Biuret reagent to each tube, mix well immediately, and incubate all the samples at room temperature for 20 min on your bench. 4. Set up the spectrophotometer. Each sample will be transferred to the cuvette for the absorbance measurement. Use the sample with no protein to calibrate the instrument. Then move on to the lowest concentration and work your way up. Read at 550 nm. The color is stable, but all readings should be taken within 10 minutes of each other. 5. Collect data for each sample, including your unknown. D. Data Analysis: 1. Open Microsoft Excel and plot your observed absorbance for each known sample as a function of protein concentration in mg/ml, 2. Examine the graphed points and decide if any should be rejected. Often a single point can be rejected without invalidating the standard curve, but if more than one point appears questionable the assay should be repeated. The Biuret assay can usually be fit to a best-fit straight line. Add a trendline (linear regression) in Excel and display the equation and display the R² value (goodness of fit). 3. Use the equation for the best-fit line in Excel to determine the protein concentration of the unknown. JIB. THE BRADFORD ASSAY Background: The Bradford dye assay is based on the equilibrium between three forms of Coomassię Blue G dye. HO₂S -S0₂ Coomassie Blue G dye Under strongly acid conditions, the dye is most stable as a doubly-pitonated red form. Upon binding to protein, however, it is most stable as an unprotonated, blue form./nL Red (470 nm) H+ Green (650 nm) H* Blue 595 nm) Blue-Protein (595 nm) The Bradford assay is fast, involves few mixing steps, does not require heating, and gives a fairly stable colorimetric. Like other assays, however, its response is prone to influence from non-protein sources, particularly detergents, and becomes progressively more non-linear at the high end of its useful protein concentration range. The response is also protein dependent and varies with the composition of the protein. These limitations make protein standard solutions necessary. Bradford Protein Assay Procedure: The objective of this assay is to collect data and plot a standard curve of absorbance vs. known concentration of proteins. Then you will determine the concentration of an unknown protein solution using the standard curve. A. Information Necessary for Data Collection. a. A spectrophotometer will be set up for you in the lab b. You will collect data at 595 nm. B. Reagents: a. Dye stock: Çoomassię Blue G (C.L.# 42655) (100 mg) is dissolved in 50 mL of methanol. (If turbid, the solution is treated with Nort (100 mg) and filtered through a glass-fiber filter.) The solution is added to 100 mL of 85% H3PO4, and diluted to 200 ml with water. The solution should be dark red, and have a pH of -0.01. The final reagent concentrations are 0.5 mg/ml Coomassię Blue G, 25% methanol, and 42.5% H3PO4. The solution is stable indefinitely in a dark bottle at 4°C. (This has been prepared for you). b. Protein Standards - Protein standards should be prepared in the same buffer as the samples to be assayed (0.1 M NaCl). A convenient standard curve can be made using bovine serum albumin with concentrations of 0, 250, 500, 1000, 1500, 2000 ug/mL for the standard assay. A 4000 µg/mL stock solution will be provided. c. Procedure: Standard Protein Assay Procedure (200- 2000 µg/mL protein concentration range): 1. Use the dilution equation to calculate the following dilutions: In 2.0-ml Eppendorf tubes, prepare 500 μL of six standard solutions containing 0, 250, 500, 1000, 1500 and 2000 µg/ml, of bovine serum albumin. Label each tube appropriately. A tube containing your unknown will be provided. This sample does not need to be diluted. Note that the tube may have less than 500 μL, in it-this is fine since you will only need to take 50 μL 2. Prepare a set of seven appropriately labeled Eppendorf tubes (ie, six for the standard solutions prepared above and one for your unknown), and to each add in sequential order: 1.5 mL Assay reagent 0.05 ml (50 μL) of protein solution, starting with the lowest protein concentration and working up. Do the unknown last. 3. Mix each tube very well. Allow them to sit for 10 minutes at room temperature on your bench top. 4. Use a plastic cuvette and calibrate the spectrophotometer at 595 nm using the sample that contains 0 Hg/ml (ie REV 0818 RB/nD. Data analysis: 1. Open Microsoft Excel and plot your observed absorbance for each known sample as a function of protein concentration. 2. Examine the graphed points and decide if any should be rejected. Often a single point can be discarded without invalidating the standard curve, but if more than one point appears questionable the assay should be repeated. The Bradford assay gives a hyperbolic plot for absorbance versus protein concentration, but within a range of relatively low protein concentrations, the hyperbolic curve can be approximated reasonably well by a straight line. Add a trendline (linear regression) in Excel and display the equation and display the R² value (goodness of fit). 3. Use the equation for the best-fit line in Excel to determine the protein concentration of the unknown. E. Preparing Your Lab Report: A full lab report should be submitted for this experiment. Refer to the "Lab Report Guidelines" document (in Blackboard) and the grading rubric for more details. General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). Data Results after the experiment: Part 1: Biuret Concentration (mg/ml) 0 1 2 5 7.5 10 Unknown A Absorbance (550nm) 0.000 0.035 0.085 0.140 0.320 0.435 0.280 REV_0818_RB/nPart 2: Bradford Concentration (µg/ml) 0 250 500 1000 1500 2000 Unknown B REV_0818_RB Absorbance (595nm) 0.000 0.134 0.315 0.717 0.987 1.074 0.549 Please use the actual concentrations of Unknown A and B given below to calculate % errors for your lab report. Unknown A: 4.8 mg/ml Unknown B: 768 pg/ml L