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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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the detection of gfp and pdi2 at different levels
The detection of GFP and PDI2 at different levels

DNA

RNA

Protein

In vivo and

In situ

ABE Workshop June 20 2006 Dongping Lu

western blotting
Western blotting

ABE Workshop June 20 2006 Dongping Lu

protein isolation
Protein isolation
  • Total protein
  • Protein in different tissues
  • Organelle protein
  • Membrane protein
  • Protein with different solubility

ABE Workshop June 20 2006 Dongping Lu

how to isolate total protein from plant
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

the often used detergents in the protein extraction buffer
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

proteases inhibitors
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

what are proteases
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

where are the proteases from when isolating the protein
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

how to prevent the proteins from degradation by protease
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

protease inhibitors
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

often used chemical protease inhibitors in protein isolation
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

the protein quantification
The protein quantification

UV 280 absorption :

Colorimetric methods:

Biuret

Lowry

Bradford

ABE Workshop June 20 2006 Dongping Lu

uv absorption method
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

disadvantages of uv absorption method
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

colorimetric methods
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

advantages of colorimetric methods
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

colorimetric methods i bradford method
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

colorimetric methods ii biuret
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

lowry method
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

advantages and disadvantages of lowry method
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

making a standard curve first with bsa
Making a standard curve first with BSA

ABE Workshop June 20 2006 Dongping Lu

today s work
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

slide24
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

the use of gfp in research
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

gfp 2sc plant
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

references
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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