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

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

3. ABE Workshop June 20 2006Dongping Lu Western blotting

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

5. ABE Workshop June 20 2006Dongping Lu 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

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

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

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

9. ABE Workshop June 20 2006Dongping Lu 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

10. ABE Workshop June 20 2006Dongping Lu 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 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 To further optimize the results, we use the proteases inhibitors

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

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

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

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

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

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

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

18. ABE Workshop June 20 2006Dongping Lu 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. 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. 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 The assay is sensitive, but somewhat non- linear

19. ABE Workshop June 20 2006Dongping Lu 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: 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 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 the absorption is relatively weak, thus, the method is somewhat insensitive and requires a relatively high concentration of protein

20. ABE Workshop June 20 2006Dongping Lu Lowry Method reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions 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 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 The Most Highly Cited Paper in Publishing History: Protein Determination by Oliver H. Lowry

21. ABE Workshop June 20 2006Dongping Lu Advantages and disadvantages of Lowry Method More sensitive than the Biuret assay (can detect lower concentrations of protein) 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). 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 More critical to timing and precision of person doing the assay

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

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

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

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

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

27. ABE Workshop June 20 2006Dongping Lu References http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html 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 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 www.bio-itworld.com/ archive/091103/russell.htmlwww.bio-itworld.com/ archive/091103/russell.htmlwww.bio-itworld.com/ archive/091103/russell.html http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo cz/gfp.htm http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo cz/gfp.htm http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo cz/gfp.htm http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo cz/gfp.htm