Lab 10- Manual Protein extractions and quantification 2 (3)

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Lab 10: Protein extraction/isolation and quantification Exercise 10.1 Choice of buffers and pigment extraction from plants. Successful preparation of crude extracts from eukaryotic cells and tissues for proteins and enzyme studies require that one pay attention to conditions that may alter the activity or native structure of an enzyme since nonspecific inactivation can result in inconsistent results and make interpretation of such studies difficult. Another point to consider is that most tissues contain various harmful agents that can degrade or harm proteins when present in a cell-free extract. Their effects need to be minimized to avoid artifacts or partially degraded proteins. This is generally accomplished by extracting at low temperatures and including various protective chemical agents (see below). Common Additions to Extraction Buffers 1. Thiol compounds are frequently added to protect proteins from oxidation. These include DTT (dithiothreitol) or 2-mercaptoethanol. 2. Chelating agents such as EDTA (ethylenediaminetetraacetic acid, often as the disodium salt) are useful in protecting enzymes from inactivation by heavy metals, which are sometimes present in reagents or released from tissue storage compartments. EDTA can prevent protein-metal ion aggregation/precipitation, substrate inhibition, or proteolysis by metalloproteases. 3. Cations are frequently added to maintain ionic strength (e.g., K + or Na + ) or provide specific stabilizing interactions (e.g., Mg 2+ ). Extraction invariably results in some instability of cellular proteins. 4. Substrates are sometimes added to stabilize enzymes and are quite specific. 5. Protease inhibitors with widely differing mechanisms and specificities are available to suppress endogenous proteases. Phenylmethylsulfonyl fluoride (PMSF) is commonly used to inactivate many serine proteases. 6. Osmotically active solutes like sucrose or sorbitol are often added to maintain the tonicity of the solution comparable to that of the cell or tissue. This way, osmotically fragile organelles like plastids and mitochondria are kept from swelling or plasmolyzing. Glycerol or other polyols are frequently added to stabilize enzymes, partly by increasing the solution viscosity. 7. Detergents are often added to solubilize organelles or membrane-associated proteins. Triton X-100 is a standard, non-ionic detergent added at 0.1-0.5%. 8. Polyvinylpolypyrrolidone (PVPP) is generally added (at 2-10% w/w) to plant extracts to prevent 'browning' from alkaloids and polyphenolic compounds such as flavonoids and

tannins. These compounds can react with and inactivate proteins by hydrogen bonding with peptide bond oxygens or by covalent modification of amino acid residues. As a general rule, foaming during extraction should be avoided as this can result in the inactivation of many enzymes through denaturation of the protein at the air/liquid interface of bubbles. Also, extractions generally are done at 4 o C to minimize proteolysis and other undesirable effects due to warming. Sometimes, extraction buffers are partially frozen to form a slurry when used. Cell Lysis Methods Bacteria are commonly lysed using a French press, as shown below, which breaks cells by pressurizing the cell suspension in a closed chamber (e.g., 7-10,000 psi) and suddenly releasing the pressure. The release of pressure creates a liquid shear capable of lysing the cells. Bacteria can also be lysed by sonication , which focuses sound waves to create a liquid shear and cavitation. The TA will demonstrate the use of a sonicator. Tissue culture cells or cell suspensions can often be lysed by a hand-held or motor-driven homogenizer , as will be shown in the lab. Solid or more fibrous tissue requires other approaches. Small samples can be extracted using a mortar and pestle . These are often frozen in liquid N 2 and then powdered before adding extraction buffer. Larger samples may require the use of a blender . A polytron homogenizer is a device with counter-rotating blades that can be useful for very fibrous tissue. Exercise 10.1 Calculate Buffer Components and Make Extraction Buffer: Materials: 1. Components of the extraction buffer 2. Spinach leaf (at TI bench) 3. Ice buckets 4. 1.5ml microfuge tubes 5. Mortar pestles 6. Centrifuge 7. Plate reader (prep room) 8. 50 ml tube with H 2 0 Determine the volumes of each of the following components to be added from the stock solutions provided in the laboratory to make 5 ml of the following extraction buffer : Keep on ice. Extraction buffer

Making Leaf Extracts 1. Put mortar pestle on the ice at the beginning of the lab to chill. 2. Make the extraction buffer using your calculations and keep it on ice. 3. Label two 1.5ml microfuge tubes CR (crude extract) and E and keep them on ice. 4. Obtain a healthy-looking spinach leaf from the TA bench. 5. Excise the leaf, remove prominent veins as feasible, and weigh 0.5 g fresh weight for each. Add weighed leaf directly to mortar. 6. Then, add 4 ml of extraction buffer and grind on ice. Grind vigorously until the leaf is homogenized (like "spinach soup"). Add one drop of extract to the tube labeled CR (crude extract) using a plastic pasture pipette . Add 50 µl of 2X SDS dye to it and leave it on ice until step 13. 7. Using the same plastic pasture pipette, transfer the extract to the microfuge tube labeled as E on ice and fill it up to 1 ml mark. 8. Working with the other team, ensure the sample tubes have equal volumes and place the balanced tubes opposite each other in a centrifuge. The rotor MUST be balanced before spinning or damage and possibly injury. Do not forget to put the lid on. 9. Spin tube E for 5 min at maximum speed. Take tubes out and place them on ice. Label a new tube as S (Supernatant ) and transfer all the Supernatant to this tube. Keep this tube Stock solutions concentrations Final concentrations Amount needed for 5ml 250 mM Tris-HCl pH 8.0 50 mM 200 mM MgCl 2 5mM 200mM EDTA 1mM In chemical hood –14.3M β-mercaptoethanol (β-ME) 10mM H 2 O (to make up to 5 ml) -

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S on ice all the time. You will need this tube in exercise 10.2 . If you discard this tube, you must start over from step # 1. 10. Put tube E with the pellet on ice and add 200 µl of extraction buffer . Flick the tube with your finger and incubate on ice for 5 mins. 11. Meanwhile, take a new tube, label it as SN, and transfer 50 µl of the Supernatant from tube S to this tube. Add 50 µl of 2X SDS dye to it and leave it on ice. 12. After 5 mins of incubation on ice, centrifuge the tube E from step 9 for 3 mins at maximum speed. Label a new tube as P (pellet) . Transfer 50µl of the Supernatant in this tube. Add 50 µl of 2X SDS dye to it and leave it on ice. 13. Take the tubes with the dye (tubes CR, SN, and P ) to the heat block on the front bench and incubate at 90°C for 5 min. 14. After 5 min of incubation, carefully take tubes out. They will be VERY HOT. 15. Relabel the tubes if needed and place the tubes in the box labeled with your section #. Your TA will store your samples at -20°C for the next lab. At the end of this exercise each team should have three tubes saved for the lab in the future.

Exercise 10.2 Bradford Protein Assay Objective: - Perform Bradford protein assay to determine the concentration of total protein extract from spinach leaves. Background Researchers need a quick and efficient way to determine the amount of protein in a solution. There are several methods established by scientists to measure protein concentrations. A suitable method is selected depending upon the choice of proteins, extraction method, feasibility, and downstream applications. The table below summarizes these methods. Method Advantages Disadvantages Example assay reagents UV absorption (280nm) Simple, doesn’t require any assay reagents Highly error prone with protein mixtures or complex samples (e.g. cell lysates) Biuret methods: Protein- copper chelation and secondary detection of reduced copper Compatibility with most surfactants (detergents) Linear response curve (R2 > 0.95) Less protein–protein variation than the Coomassie dye–based assays Incompatibility with substances that reduce copper Incompatibility with common reducing agents such as DTT BCA Assays Lowry Assays Colorimetric dye based methods: Protein-dye binding and direct detection of the color change Fast and easy to perform Performed at room temperature Compatible with most salts, solvents, buffers, thiols, reducing substances, and metal-chelating agents Incompatibility with surfactants (detergents) High protein–protein variation when compared to copper-based assays Bradford (Coomassie) Fluorescent dye methods: Protein-dye binding and direct detection of increase in fluorescence associated with the bound dye Excellent sensitivity, requiring less protein sample for quantitation Timing is not a critical factor, so the assays can be adapted for automated handling in high- throughput applications Requires specialized equipment EZQ fluorescent assay Qubit Protein Assay Since the Bradford assay is easy to perform in the labs, we will select Bradford as a method for quantifying total protein from spinach leaf extract. The Bradford protein assay (commercially available through Bio-Rad, Hercules, CA) is a dye-binding assay in which a color change (light brown to blue) occurs upon the binding of Coomassie Blue G-250 dye to protein as shown in the figure below. The higher the protein concentration in a solution, the more dye binds and the greater the color change, hence greater absorbance. Figure 10.1 Reaction schematic for the Bradford assays

How does this absorbance relate to the actual protein concentration? A standard curve is required to determine the actual concentration of a protein. A standard curve is a plot of absorbance vs. a varying amount of known protein that you want to know the concentration of. A common method to prepare a standard curve with known protein concentrations as standards. As long as the volume of the standard samples and the unknown samples are the same, the final concentration of the unknown is directly calculated from the least squares line of the standard curve. Of course, you have to correct for any dilution of your sample. The assay is linear only in a specific range ( 0.05 – 0.5 mg/ml) . The addition of more protein will not result in any additional color change. Conversely, very low concentrations of proteins will not result in measurable color changes. Sample Preparation: When determining the protein concentration of an unknown sample, several dilutions should be used to ensure the protein concentration is within the range of the assay. Usually, 2- and 10-fold dilutions are used to get the unknowns within the standard curve range. Don't forget that all dilutions must be considered when calculating the protein's final concentration. Think about the dilution factors. Finally, with every assay, a "blank" must be included. The "blank" is used to set the instrument to 0 absorbance. The blank or the tube without a protein is usually made up of the same buffer as in the samples. Exercise 10.2 Bradford Protein Assay Materials: - Protein extract of unknown concentration ( Tube S with Supernatant from exercise 10.2) - Protein Assay Dye Reagent (on TA bench, in ice, will be distributed after checking the calculations) - BSA (Bovine Serum Albumin): 1 mg/ml in deionized water; stored at -20 o C (This is a protein solution of known concentration for building a standard curve). - H 2 O (for making dilutions of protein standard and unknown protein samples) - 96-well microplate, 1 per 2 students - Thermo Scientific Multiscan FC microplate photometer (See Appendix for more detail)

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Procedure: A protein assay consists of two main components : 1. building the standard curve 2. calculating the concentrations of the unknown protein extracts. I. Setting up the assay and taking measurements 1. Each team of 2 students will perform 1 Bradford assay : 1 standard curve and unknown sample with dilutions of the extract from tube S. 2. You will need to prepare a series of BSA standard solutions of known concentrations that would allow you to build a standard curve. The standard curve is a plot of the light absorbed by the proteins vs its corresponding concentration. (You want to have at least 5 standard concentrations within the linear range of the assay + a blank sample). Because you don't know if the protein concentration of your "Unknown" plant extract from tube S, is within the range of your standard curve, you might want to prepare a series of the unknown protein extracts (for example, a 2- fold or 10-fold dilutions). 3. Use the table below to calculate the volume of the BSA standard (1 mg/ml) and water you need to prepare each protein standard; and volume of the unknown protein extract (PE-1 to PE-3) and water to prepare 2-fold, 10-fold, and 20-fold dilutions of the unknown proteins. You can prepare enough protein solutions for all three reps for standards and the unknowns. (To simplify the math, we'll prepare 100 µl of the protein solution for each standard and unknown protein). 4. Label Nine 1.5mL tubes and prepare 100 µl of each standard and unknown protein dilutions. Review the table before labeling. Ask your TA/TI to check your math before you start pipetting! Tube # Sample Protein conc mg/mL µL of BSA or Unknown V of H 2 O, µL 1 Blank 0 0 100 2 Standard 1 0.05 5 95 3 Standard 2 0.1 4 Standard 3 0.2 5 Standard 4 0.3 6 Standard 5 0.4 7 (PE-1) Unknown Supernatant from tube S 2-fold dilution ? 8 (PE-2) Supernatant with 10-fold dilution ? 9 (PE-3) Supernatant with 20-fold dilution ?

5. Flick the tubes to mix the protein solutions. 6. For Bradford assay performed in 96-well microtiter plate format, you will use; 10 µl of protein solution to wells 1-6 (or water, if it's a blank sample) and 10 µl of diluted extracts to wells 7-9 . 6.1. The upper-left corner well is used for a blank sample (no protein). (See Figure 10.4 on the next page). We recommend that the "standard" samples and the "unknown" samples are done in triplicates to reduce the effects of the pipetting error. Standards PE-1 PE-2 PE-3 Figure 10.4. Bradford Assay plate layout 7. Add 10 µl of the respective protein sample in the corresponding well in triplicates. Column 1A: H 2 0 (Blank) Column 2-6: Standards 1-5 in triplicates Column 7: Unk1 2-fold dilution of extract from tube S in triplicates Column 8: Unk1 10-fold diluted of extract from tube S in triplicates Column 9: Unk1 20-fold diluted of extract from tube S in triplicates 8. Add 190 µl of Protein assay dye (from the dark tube) to each of the above samples, using a P-200 pipette. 9. Once all the samples are in the well, make sure there are no air bubbles. 10. Measure the light absorbency at 595 nm in a microtiter plate reader (Your TA will perform this step for you and provide you with a printout of the data). The measurements need to be taken within 5-60 min. after mixing the dye with the protein solution. 11. If you need to repeat the assay, you can use empty rows on the same plate.

12. Take a picture of your raw data from the plate reader. Clean-up 1. Wash the tube used for making buffer and put it back on your bench. 2. Wash mortar pestle and put it back on your bench. 3. Dump the contents of the 96-well plate in the liquid waste in fume hood and put the plate in trash. 4. Put the 9 tubes with standards and dilutions in trash. Save: 1. Return the tube with 2X SDS-Dye and BSA to your TA/TI. 2. Leave MgCl 2 , EDTA, Tris-HCl and H 2 0 on the bench. We will reuse it for next lab sections. II. Data analysis 1. Transfer your data to Excel file. 2. Calculate absorbance values for each sample by subtracting the Blank reading from every sample reading. 3. Calculate average for every standard and every unknown sample. (Remember, you made 3 replicates for each standard and unknown?) 4. Make a standard curve by graphing the independent variable (series of BSA standard concentrations, mg/ml) on the X-axis and the dependent variable (abs 595 nm) on the Y- axis. Example: Concentrations(mg/ml ) 0.05 0.1 0.2 0.3 0.4 Average absorbance (A 595 ) 0.181 0.313 0.447 0.544 0.609 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Concentrations (mg/ml) Absorbance (595 nm)

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5. Go to the layout and display the standard curve, the equation, and the R 2 . 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 f(x) = 1.18 x + 0.17 R² = 0.95 Concentrations (mg/ml) Absorbance (595 nm) The closer to 1.0 your "R 2 " value is, the more in line your data are. 0.9 to 1.0 are reasonable values. 6. Use linear regression to calculate the concentration in your unknown samples. If you dilute your sample, don't forget to multiply the concentration by your dilution factor. 7. Use the calculated average values of your unknown samples and plug in the equation. Note: you should plug in your absorbance for unknown for Y and calculate X for each dilution. 8. Discuss if the calculated values for each dilution are relatively closer to each other. If not what could have contributed to this discrepancy. Reference Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry , 72, 248-254 Lab Report: (Includes lab 9 and 10) The lab report for lab 9 and lab 10 are combined and submitted as one. You will perform protein separation next week and submit the lab report before the end of the following week. We suggest you complete part of the lab report for lab 9 this week to stay on track.