Transcript Genetically Modified Organism

ABE Workshop 2007
Protein Isolation and
Quantification
DNA
RNA
Protein
How to isolate total protein
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Lyse the cell,
Solubilize the proteins:
To
Solubilize
membrane
protein, we have to use
detergents in the protein
extraction buffer
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
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?
What are proteases?
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Protease: (proteinases, peptidases or
proteolytic enzymes) are enzymes that break
peptide bonds between amino acids of proteins
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?
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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
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
The protein quantification
UV 280 absorption :
Colorimetric methods:
Biuret
Lowry
Bradford
UV absorption method
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The amino acids tryptophan, tyrosine and
phenylalanine absorb light in the UV wavelength
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Since the absorption is proportional to
concentration, this is a useful way to quantitates
protein concentration (for proteins containing Trp)
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,
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.
Advantages of Colorimetric methods
1. Cheap cuvette! (cheap glass or plastic versus
quartz quartz)
2. Not contaminating absorbance from nucleic
acids!
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.
This dye (which normally absorbs at 465nm)
binds to proteins and to absorb strongly at
595nm.
The assay is sensitive, but somewhat nonlinear
Lowry Method
A widely-used method of
measuring protein
concentration
A colorimetric assay
Amount of blue color
proportional to amount of
protein
Absorbance read using 500750nm light
Lowry et al, 1951
Lowry Method
Two reactions make the blue color
develop:
 Reaction 1
Cu2+ + peptide bonds → Cu1+-peptide
bond complex, produces purple-blue color
 Reaction 2
Folin reagent + Cu1+-complex → reduced
Folin reagent, produces blue-green
Making a standard curve
with BSA (bovine serum albumin)
A graph that correlates Absorbance with protein
concentration
Standard Curve generated by doing a Lowry Assay
on protein solutions of known concentration
Standard Curve must be done each time unknowns
are being tested
The SDS-PAGE
PAGE
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Gels are cast by polymerizing a solution of acrylamide
monomers into polyacrylamide chains
Gel pore size can be varied by adjusting the concentrations
of polyacrylamide
Smaller proteins migrate faster than larger proteins through
the gel
Native proteins
SDS (sodium dodecyl sulfate) binds to and
coat the protein
SDS
1. SDS disrupts some of the noncovalent
interactions that stabilize protein
quaternary and tertiary structures,
facilitates denaturation.
2. SDS also has a negative electrical
charge and binds to proteins in a constant
mass ratio of 1.4 : 1, so that the total
amount of detergent bound is directly
proportional to the molecular weight of
the protein.
3. The ‘coating’ of negatively charged
SDS overwhelms the inherent charges of
protein molecules and gives them a
uniform charge to mass ratio.
4. This allows proteins to be separated on
the basis of their relative sizes,
SDS
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all polypeptide chains are then forced into extended
conformations
SDS treatment eliminates the effect of differences in
shape
individual polypeptide chains migrate as a negatively
charged SDS-protein complex through the porous
polyacrylamide gel
speed of migration is proportional to the size of the proteins
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smaller polypeptides running faster than larger polypeptides
How about covalent link?
DTT/Me
SH
S-S
HS
Noncovalent
covalent
Heating the sample
Heating your samples at 99ºC completed denaturation of
the protein molecules, ensuring that they were in completely
linear form.
 This allowed SDS to bind all regions of each protein
equally.
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Protein loading buffer
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Protein gel loading buffer contains Tris buffer to
maintain constant pH
glycerol to increase sample density,
the strong ionic detergent SDS (sodium
dodecylsulfate),
β-mercaptoethanol, a reducing agent. . Betamercaptoethanol eliminates disulfide bonds in
proteins by reducing them (adding hydrogen
atoms).
Heating
Running the gel
Stacking gel
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To obtain optimal resolution of proteins, a “stacking” gel is
poured over the top of the “resolving” gel.
The stacking gel
lower concentration of acrylamide (larger pore size),
lower pH
different ionic content
This allows the proteins in a lane to be concentrated into a
tight band before entering the running or resolving gel
produces a gel with tighter or better separated protein
bands
Gel staining
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Once proteins have been fractionated by
electrophoresis, to make them visible, staining with
a material that will bind to proteins but not
polyacrylamide.
the most common one: staining with Coomassie
Blue.
This is a dye that binds most proteins uniformly
based on interactions with the carbon-nitrogen
backbone.
The dye is dissolved in a solution that contains both
methanol and acetic acid
gel-drying frames
for drying of SDS-PAGE gels
Gel drying
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SDS-PAGE gels between two moistened
sheets of Gel Drying Film (from Promega)
on the bench.
Clamp the Gel Drying Frame
Dry over night
It is important to remove all the air bubbles
from between the two sheets of gel drying
films. Air bubbles may cause the gel to
crack during drying
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
Transfer
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In this procedure, a sandwich of
gel and solid support membrane
(Nitrocellulose or PVDF) is
compressed in a cassette and
immersed in buffer between two
parallel electrodes.
A current is passed at right angles
to the gel, which causes the
separated
proteins
to
electrophorese out of the gel and
onto the solid support membrane
Transfer the protein from the gel to the
membrane
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Transfer of the proteins fractionated by SDSPAGE to a solid support membrane (Western
blotting) can be accomplished by electroblotting