Isolation and quantification of plant total protein

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Isolation and quantification of plant total
protein
ABE Workshop 2006

• Dongping Lu

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The detection of GFP and PDI2 at different levels
DNA
RNA
Protein
In vivo and In situ
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Western blotting
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Protein isolation

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

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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

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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

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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?

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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
• Aspartate proteases
• Cysteine proteases

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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

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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

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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

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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

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The protein quantification

• UV 280 absorption
• Colorimetric methods
• Biuret
• Lowry
• Bradford

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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)

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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,

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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.

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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!

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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

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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

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Lowry Method

• reactivity of the peptide nitrogens 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
  

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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

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Making a standard curve first with BSA
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Todays work

• Isolate the total protein
• group 1 4 wt and pdi2 mutant plant
• group 2 3 wt and gfp-2sc plant

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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.

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The use of GFP in research

• 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.

PDI
GFP
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GFP-2SC Plant
SP the signal peptide of pumpkin 2S albumin
2SC Vacuolar-targeting signals of pumpkin 2S
albumin
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References

• http//www.bio.davidson.edu/people/jowilliamson/Te
  chniques/Protocolweek5.html
• Lowry, O. H., Rosebrough, N. J., Farr, A. L., and
  Randall, R. J. (1951) J. Biol. Chem.193, 265275
• 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