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Isolation and quantification of plant total protein

ABE Workshop 2006. Isolation and quantification of plant total protein. Dongping Lu. The detection of GFP and PDI2 at different levels. DNA. RNA. Protein. In vivo and In situ. Western blotting. Protein isolation. Total protein Protein in different tissues Organelle protein

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Isolation and quantification of plant total protein

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  1. ABE Workshop 2006 Isolation and quantification of plant total protein Dongping Lu

  2. The detection of GFP and PDI2 at different levels DNA RNA Protein In vivo and In situ ABE Workshop June 20 2006 Dongping Lu

  3. Western blotting ABE Workshop June 20 2006 Dongping Lu

  4. Protein isolation • Total protein • Protein in different tissues • Organelle protein • Membrane protein • Protein with different solubility ABE Workshop June 20 2006 Dongping Lu

  5. How to isolate total protein from plant • Lyse the plant cell, • Solubilze the proteins: • To Solubilze membrane protein, we have to use detergents in the protein extraction buffer ABE Workshop June 20 2006 Dongping Lu

  6. The often used detergents in the protein extraction buffer • Nonionic detergents(milder) Triton X-100: break lipid-lipid interaction and lipid-protein interaction • Anionic detergents (more denaturing) SDS: protein-protein interaction Sodium Deoxycholate: protein-protein interaction ABE Workshop June 20 2006 Dongping Lu

  7. Proteases inhibitors • Upon lysis of the cell, proteases are released into the lysate • What are proteases? • Where are the proteases from when isolating the protein? ABE Workshop June 20 2006 Dongping Lu

  8. What are proteases? • Protease: (proteinases, peptidases or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins • Classes of proteolytic enzymes: Serine proteases Aspartateproteases Cysteine proteases ABE Workshop June 20 2006 Dongping Lu

  9. Where are the proteases from when isolating the protein? • Animal cells: Lysosomes, contain a large variety of hydrolytic enzymes that degrade proteins and other substances • Plant cells: Vacuole, many hydrolytic enzymes found in vacuole resemble those present in Lysosomes of animal cells other organelles also have proteases ABE Workshop June 20 2006 Dongping Lu

  10. How to prevent the proteins from degradation by protease? • the protein isolation is carried out at low temperature to minimize the activities of these proteases • To further optimize the results, we use the proteases inhibitors ABE Workshop June 20 2006 Dongping Lu

  11. Protease inhibitors • Proteins: with domains that enter or block a protease active site to prevent substrate access, e.g. Cystatins. • Chemicals: some are used in the protein extraction buffer ABE Workshop June 20 2006 Dongping Lu

  12. Often used chemical protease inhibitors in protein isolation • EDTA (or EGTA): chelating the Ca2+, • PMSF: a general serine protease inhibitor. It is the most common inhibitor used in protein purification. Soluble in isopropanol. • The protease inhibitors cocktail: a mixture of several protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic and aminopeptidases ABE Workshop June 20 2006 Dongping Lu

  13. The protein quantification UV 280 absorption : Colorimetric methods: Biuret Lowry Bradford ABE Workshop June 20 2006 Dongping Lu

  14. UV absorption method • The amino acids tryptophan, tyrosine and phenylalanine absorb light in the UV wavelength • Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp) ABE Workshop June 20 2006 Dongping Lu

  15. Disadvantages of UV absorption method • If some proteins do not contain these amino acids, it will not absorb UV light, • Nucleic acids (DNA, RNA) contaminant will also absorb UV light, ABE Workshop June 20 2006 Dongping Lu

  16. Colorimetric methods • we can modify the protein sample with appropriate reagents so as to produce a color reaction and measure protein concentration using a spectrophotometer. ABE Workshop June 20 2006 Dongping Lu

  17. Advantages of Colorimetric methods 1. Cheap lamp! (tungsten light bulb versus deuterium for UV) 2. Cheap cuvette! (cheap glass or plastic versus quartz) 3. Not contaminating absorbance from nucleic acids! ABE Workshop June 20 2006 Dongping Lu

  18. Colorimetric methods I: Bradford Method • A dye known as Coomassie Brilliant Blue was developed by the textile industry. It was noticed to stain skin as well as the textiles. • Thus, this dye (which normally absorbs at 465nm) was known to bind to proteins and to absorb strongly at 595nm. • The assay is sensitive, but somewhat non-linear ABE Workshop June 20 2006 Dongping Lu

  19. Colorimetric methods II: Biuret • Under high pH (alkaline) conditions the copper II ion (Cu2+) is believed to form a complex with peptide nitrogens of proteins: • This complex absorbs light at 550nm • the absorption is relatively weak, thus, the method is somewhat insensitive and requires a relatively high concentration of protein ABE Workshop June 20 2006 Dongping Lu

  20. Lowry Method • reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions • reduction of Folin-Ciocalteu reagent, resulting in a color change from yellow to blue, which absorbs strongly at 750nm • The Most Highly Cited Paper in Publishing History: Protein Determination by Oliver H. Lowry ABE Workshop June 20 2006 Dongping Lu

  21. Advantages and disadvantages of Lowry Method • More sensitive than the Biuret assay (can detect lower concentrations of protein) • Absorption reaction is linearly dependent upon protein concentration, but only at low concentrations of protein (i.e. the standard curve and assay must be performed at a low concentration regime). • More critical to timing and precision of person doing the assay ABE Workshop June 20 2006 Dongping Lu

  22. Making a standard curve first with BSA ABE Workshop June 20 2006 Dongping Lu

  23. Today’s work • Isolate the total protein: group 1 & 4: wt and pdi2 mutant plant group 2 & 3: wt and gfp-2sc plant ABE Workshop June 20 2006 Dongping Lu

  24. GFP • Green Fluorescent Protein: a fluorescent protein isolated from jellyfish. • Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. ABE Workshop June 20 2006 Dongping Lu

  25. The use of GFP in research PDI GFP • The gene for GFP has been isolated • It has become a fluorescent protein tag to makeing chimeric proteins • It has been expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish, and in mammalian cells. ABE Workshop June 20 2006 Dongping Lu

  26. GFP-2SC Plant SP: the signal peptide of pumpkin 2S albumin 2SC: Vacuolar-targeting signals of pumpkin 2S albumin ABE Workshop June 20 2006 Dongping Lu

  27. References • http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html • Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem.193, 265–275 • www.bio-itworld.com/ archive/091103/russell.html • http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.htm ABE Workshop June 20 2006 Dongping Lu

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