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

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Dec 6, 2023

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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) -

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.

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