Transcript Genetically Modified Organism

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
ABE Workshop
June 20 2006
Dongping Lu
Western blotting
ABE Workshop
June 20 2006
Dongping Lu
Protein isolation
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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
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ABE Workshop
Lyse the plant cell,
Solubilze the proteins:
To
Solubilze
membrane
protein, we have to use
detergents in the protein
extraction buffer
June 20 2006
Dongping Lu
The often used detergents in the protein
extraction buffer
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Nonionic detergents (milder)
Triton X-100: break lipid-lipid interaction and
lipid-protein interaction
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Anionic detergents (more denaturing)
SDS: protein-protein interaction
Sodium Deoxycholate: protein-protein interaction
ABE Workshop
June 20 2006
Dongping Lu
Proteases inhibitors
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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?
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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
ABE Workshop
June 20 2006
Dongping 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
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ABE Workshop
June 20 2006
Dongping Lu
How to prevent the proteins from
degradation by protease?
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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
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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
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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 ABE
andWorkshop
aminopeptidases
June 20 2006
Dongping Lu
The protein quantification
UV 280 absorption :
Colorimetric methods:
Biuret
Lowry
Bradford
ABE Workshop
June 20 2006
Dongping Lu
UV absorption method
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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
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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
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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
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
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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 nonlinear
ABE Workshop
June 20 2006
Dongping Lu
Colorimetric methods II: Biuret
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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
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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
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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
ABE Workshop
June 20 2006
Dongping Lu
Today’s work
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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
GFP
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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
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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
PDI
Dongping Lu
GFP
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
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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/gmo
cz/gfp.htm
ABE Workshop
June 20 2006
Dongping Lu