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Protein isolation and quantification

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Protein isolation and quantification

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Protein isolation and quantification

  1. 1. .
  2. 2. Protein Isolation and Quantification BY : JAYANT YADAV, CCSHAU, HISAR, HARYANA
  3. 3. Protein extraction Protein Proteins are the biomolecules, composed of amino acid, forming the building block of the system and performs most of the biological functions of the system. Protein extraction: The process by which the proteins from the cell are recovered for the analysis purpose is called protein extraction
  4. 4. Why ? Proteins are extracted from tissues for a wide range of reasons, including  To compare the structure of proteins as expressed by different organism.  To purify a protein in order to identify the gene that encodes it, and  To resolve proteins by SDS-PAGE.  To assay an enzyme in a crude extract for physiological studies.  To study the mechanism of action of an enzyme.  To diagnose parasitic diseases.
  5. 5. Generic outline for protein isolation 1. Collection of sample, washing, cleaning and grinding to fine powder 2. Cell lysis with physical (Grinding) and chemical (lysis buffers) methods. 3. Separation of proteins from non-protein components (nucleic acids and lipids) & recovery of bulk proteins from a crude extract 4. Further separation of target protein-containing fraction from the bulk proteins 5. Recovery of the target protein in a highly purified state along with a high yield
  6. 6. Materials  Frozen or fresh plant material  Liquid nitrogen  Extraction buffer  Mortar & pestle or tissue grinder  20- to 70-μm pore size nylon mesh  Refrigerated centrifuge and centrifuge tubes Extraction of Proteins from Plant Tissues
  7. 7. procedure Cleaning and weighing of Leaves  Take isolated leaves in a petri-plate & wash with distilled water to remove all the particulate matter on the surface.  Dry the leaves on a tissue paper without applying excess pressure.  After the leaves are totally dry, weigh the required amount on a balance.
  8. 8. Liquid Nitrogen Treatment  Take a clean mortar and pestle and place it on ice.  Pre-chill the mortar with liquid nitrogen.  Transfer the leaves into the pre-chilled mortar & Add around 10 ml of liquid nitrogen, all at one go.
  9. 9. Grinding the Leaves & Lysis Buffer Treatment  Grind the leaves till a fine powder is seen.  To the powdered leaves, add lysis buffer and grind thoroughly.  Transfer the lysate to fresh eppendorf tubes & Add some more lysis buffer, to ensure proper lysis of cells.  Vortex the tube thoroughly to mix the contents uniformly. Protein Precipitation at -20°C  After vortexing the contents, incubate the tube at -20°C for 1 hour.  The proteins at the end of this incubation, get precipitated in the buffered solution.
  10. 10. Centrifugation and Acetone Wash  Take the tube out of -20oC freezer. Centrifuge the tube at 14000 rpm, 4oC for 30 minutes,  Take the tube out of the centrifuge. The tube can be seen to have a clearly demarcated pellet and supernatant. Discard the supernatant carefully without disturbing the pellet.
  11. 11.  To the pellet, add 1ml of wash buffer. Wash buffer helps in removing the color of the pellet.  Vortex the tube to thoroughly let the pellet dissolve into the buffer. Incubate the tube in the -20o freezer for 30 minutes.  Remove the tube from the freezer carefully. Centrifuge the tube at 14000rpm, 4oC for 15 minutes.  Discard the supernatant carefully without disturbing the pellet. Air dry the pellet to remove even traces of acetone.
  12. 12. Rehydration Buffer Treatment  Add 400 μL of rehydration buffer to the air-dried pellet.  Vortex the tube till the pellet dissolves in the rehydration buffer (CHAPS solubilizes the proteins, Urea denatures the proteins.)  Incubate the tube overnight at 4oC for complete solubilisation of proteins in the rehydration buffer. Sample Storage  The sample contains the protein  If not required immediately it can be stored at -20°C for later use.
  13. 13. Protein isolation from animal tissues Materials  Waring blender  Refrigerated centrifuge  Polypropylene screw-cap centrifuge tubes
  14. 14. Procedure 1. Trim fat and connective tissue from the body and cut into small pieces 2. Place the tissue in the precooled blender vessel and add cold extraction buffer(2) 3. Homogenize at full speed for 1—3 min depending on the toughness of the tissue. 4. Pour the homogenate into a glass beaker, place on ice, and stir for 15-30 min to ensure full extraction.
  15. 15. 5. Remove cell debis and other particulate matter from the homogenate by centrifugation at 4°C. 6. Pour off the supernatant carefully to avoid disturbing sedimented material. Any fatty material that has floated to the top of the tube should be removed by filtering the extract through cheese cloth 7. The pellet may be re-extracted with more buffer to increase the yield. 8. The filtrate obtained at this stage may require further treatment to remove insoluble material 9. Thus obtained is protein extract.
  16. 16. Protein extraction from fungi Materials  Malt extract agar (MEA)  Glucose yeast medium (GYM)  Pectin broth  Tris-glycine buffer  Pectinase gel  Gel buffer  Electrode buffer  0.1 Malic acid
  17. 17. procedure This protocol describes the extraction of aqueous intracellular proteins from the filamentous fungus Metarhizium anisopliae  Harvest the mycelium from given culture by vacuum-assisted filtration onto Whatman No. 3 filter paper.  Wash the mycelium once in the Buchner funnel with sterile deionized water, and transfer the harvested mycelium from the filter paper to a plastic Petri dish with a sterilized spatula.  Freeze the mycelium at -20°C for 24 h.
  18. 18.  Disrupt freeze dried mycelium by briefly grinding it in a mortar and pestle & collect it in sterile 1.5 mL micro-centrifuge tubes (~500-mg). Rehydrate it in 1 mL of Tris-glycine buffer.  Clarify the slurry by centrifugation at 12,500 g for 40 min at 4°C. After centrifugation, collect the supernatant into another sterile micro-centrifuge tube  The collected supernatant will contain the total cytoplasmic proteins. The samples as prepared here typically contain 15-100 mg/mL protein
  19. 19. Protein extraction form Bacteria Materials  Lysis buffer  Hen egg lysozyme  DNase 1  Sodium deoxycholate  MgCl2
  20. 20. procedure 1. Harvest the bacterial cells by centrifugation at 1000g for 15 min at 4°C, and pour off the supernatant. 2. Weigh the wet pellet. 3. Add approx 3 mL of lysis buffer for each wet gram of bacterial cell pellet and resuspend. 4. Add lysozyme to a concentration of 300 µg/mL and stir the suspension for 30 min at 4°C
  21. 21. 5. Add deoxycholate to a concentration of 1 mg/mL while stirring. 6. Place at room temperature, and add DNase 1 to a concentration of 10 mg/mL and MgCl2 to 10 mM. Stir suspension for a further 15 min to remove the viscous nucleic acid 7. Centrifuge the suspension at 10,000g for 15 min at 4°C. 8. Resuspend the pellet in lysis buffer to the same volume as the supernatant, and analyze aliquots of both for the protein of interest on SDS-PAGE
  22. 22. SDS-PAGE analysis SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis  It is a common technique used to separate, visualize, and therefore compare the relative amount of individual polypeptide chains contained in different fractions  It yields the mass of each individual subunit derived from the denatured complex
  23. 23. procedure Denaturation:  A small sample from each fraction is first mixed with an excess of SDS, ßME and bromophenol blue.  Each mixture is then placed in a boiling water bath for several minutes  In the end, each polypeptide chain is unfolded into a negatively- charged, rod-shaped complex with a relatively constant charge to mass ratio.
  24. 24. Electrophoresis:  Take a solid, rectangular gel of polyacrylamide that is cross- linked to a desired mesh or size exclusion limit.  A small aliquot from each denatured sample is transferred to separate wells of gel.  An electric field is applied across the loaded gel with the positive pole positioned on the opposite side of the samples
  25. 25.  Negatively-charged complexes get drived the through the mesh of polyacrylamide which effectively filters them according to the length of their Stokes radius as they wiggle through the porous matrix to the opposite side of the gel.  Small particles move through the gel more rapidly than larger ones,  The electrophoresis is visually followed. Power supply is shut off before any protein in the sample reaches the bottom of the gel.
  26. 26. Staining and destaining:  Following electrophoresis, the entire gel is soaked in a stain solution containing a dye that tightly binds to the backbone of each denatured polypeptide chain (usually Coomassie brilliant blue).  Excess stain is then washed from the gel by soaking it in a destain solution long enough for the blue-stained bands of each polypeptide to be visualized against the clear, colorless background of the gel.
  27. 27. PAGE of a sample proteins stained with Coomassie blue Two SDS-PAGE-gels after a completed run
  28. 28.  After staining, different species biomolecules appear as distinct bands within the gel.  It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel  Approximate molecular mass of unknown biomolecules is determined by comparing the distance traveled relative to the marker. Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane
  29. 29. protein concentration by spectrophotometer  This method is recommended for pure protein solutions Principle  Proteins in solution absorb UV light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm.
  30. 30. Procedure 1. Warm up the UV lamp (about 15 min.) 2. Adjust wavelength to 280 nm 3. Calibrate to zero absorbance with buffer solution only 4. Measure absorbance of the protein solution 5. Adjust wavelength to 260 nm 6. Calibrate to zero absorbance with buffer solution only 7. Measure absorbance of the protein solution
  31. 31. Analysis  Unknown proteins or protein mixtures. Concentration(mg/ml) = Absorbance at 280 nm path length (cm.)  Pure protein of known absorbance coefficient: for 1cm path length concentration = Absorbance at 280 nm absorbance coefficient
  32. 32. Concentration may in mg/ml, %, or molarity depending on type coefficient is used. Mg protein/ml = % protein 10 = molarity Protein Molecular weight  Unknowns protein with possible nucleic acid contamination. Conc. (mg/ml) = (1.55 x A280) - 0.76 x A260)
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