Protein Detection Methods & Western
Blots
1. Sodium Dodecyl Slufate (SDS)-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
• Visualize many proteins at once
2. SDS-PAGE combined with immunoblotting
(Western)
• Visualize one protein out of complex mixture
What is SDS-PAGE?
A procedure to separate proteins and determine their Molecular
Weights.
Theory Behind Electrophoresis
• Charged molecules in an electric field behave in a
predictable manner.
• Positively charged molecules will move towards the
negative pole while negatively charged molecules move
towards the positive pole.
• Movement of any charged species through an electric
field is determined by it’s net charge, molecular radius
and the magnitude of the applied field, but not size
How do we get proteins to separate by
size?
•Need to disrupt tertiary structure
•Make charge uniform
The importance of SDS
SDS is a negatively charged (anionic) detergent.
Coats proteins and disrupts secondary and tertiary protein
structures by breaking hydrogen bonds and unfolding or
denaturing protein.
‘Masks’ charge on protein by imparting a large negative
charge so that all proteins act the same in regards to charge.
Prevents protein aggregation.
Prevents protein shape from influencing gel run. Size of
protein determines migration in gel.
SDS linearizes proteins
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•All proteins have the same mass to charge ratio
•Have the same molecular radius
•Thus, migration through a matrix is determined
by length which is proportional to molecular
weight
SDS-PAGE: The Matrix
Free radicals
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Transfer of electrons to acrylamide and
bisacrylamide; causing them to react
Amount of crosslinking, thus pore size and
consequent separation properties of the
gel can be controlled by varying the ratio
of acrylamide to bis-acrylamide
SDS-PAGE: The buffer system
Discontinuous Laemmli buffer system buffer in the gel and the tank are different
Gly
proteins
Cl-
Stacking gel: 4%
Resolving gel: range 5-20%
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Proteins are concentrated into the narrow
zone between the Cl- and glycine fronts.
Polyacrylamide concentration alters
resolution of protein migration
Uses of SDS-PAGE
•
•
•
•
•
Determine protein size
Identify protein
Determine sample purity
Identify existence of disulfide bonds
Quantify amounts of protein
Detection of protein after SDS-PAGE
STAINING
Technique - The goal of staining is to bind a chromaphore to a
polypeptide
Not Quantitative - All stains are very qualitative, individual polypeptides
differ greatly in their ability to bind a stain
Low Reproducibility - Different staining techniques will not stain a
polypeptide consistently
Range - There is a small concentration range over which a particular
stains is useful
Non-Specific – Binds to majority of proteins; unable to determine if your
protein of interest is in the protein extract run on the gel
Protein dyes used for staining
Stain
Coomassie
Blue
Advantages
Disadvantages
Simple methodology
MS compatible
Easy to image
Very insensitive
Bio-Safe
Colloidal
Coomassie
Stain
Simple methodology
MS compatible
More sensitive
Easy to image
Less sensitive than
silver
Silver stain
Very sensitive
Easy to image
Complex procedure
Incompatible with MS
Not quantitative
Sliver stain
Plus
Very sensitive
Easy to image
More MS compatible
than standard silver
staining
Very sensitive
MS compatible
Quantitative over 3
orders of magnitude
Simple methodology
Complex procedure
Not very MS
compatible
Not quantitative
Ruby stain
Difficult to image
Other Methods of Protein Detection
1. Labeling proteins with radioisotopes during
expression (e.g. 35S-methionine)
1. Western blotting (e.g. antibody against protein
of interest)
Bio-Rad’s take on Western blotting!
Comparative Proteomics Kit II:
Western Blot Module
According to Bio-Rad
• Powerful teaching tool
Why Teach
Western Blotting?
• Real-world connections (?)
• Laboratory extensions (?)
• Tangible results
• Link to careers and industry (?)
• Standards-based (?)
Why use Western blotting?
•Can specifically determine if your protein of
interest is in present in a crude protein
preparation
Transfer
Proteins from
the gel to the
nitrocellulose
membrane
Blotting buffer
1x Tris glycine
with 20%
methanol,
0.1% SDS
Electric Current
60 minutes
200 mA
Protein Transfer
Blocking membrane before addition of antibody
Remove membrane from the blotting
sandwich and immerse in blocking
solution
Blocking
Buffer
5% non-fat milk or 1% gelatin: Prevents
the primary antibody from binding nonspecifically to the membrane
Tris or Phosphate buffered saline (TBS or
PBS): Provides the correct environment
(pH, Salt) to maintain protein shape
0.025% Tween 20: non-ionic detergent
that prevents non-specific binding of
antibodies to the membrane; TBS-T or
PBS-T when added to buffer
Antibodies used for Western
• Molecule of interest is injected into
primary animal model
• Animal makes antibodies against the
molecule
• Antibodies are purified (primary antibody)
Addition of the
o
1
antibody
•Discard blocking solution
•Pour primary antibody (in TBST) onto the
membrane and gently rock for 30 minutes
•Primary antibody will bind to the histidine
tag
•Add 50ml of wash (TBST) rock for 10 min to
wash
Addition of
o
2
Antibody
•Discard wash solution
•Pour secondary antibody onto the
membrane and gently rock for 30 minutes
•Secondary antibody will bind to the primary
antibody
•Quickly rinse membrane in 50ml of wash
buffer and discard the wash buffer
•Add 50ml of wash leave for 3 minutes on the
rocking platform
Production of
o
2
Antibody
• Antibodies from the first animal model are
injected into a second animal model
• The second animal produces antibodies
against the first antibody (secondary
antibody)
• The secondary antibody is purified and
conjugated to a colorimetric substrate or
to an enzyme that can cleave a
colorimetric compound
Detection of Protein
•Discard wash solution
•Add 10ml of the enzyme substrate (HRP color
detection reagent) onto the membrane
•Incubate for 10 minutes
•The colorimetric substrate is cleaved by the
enzyme conjugated (attached) to the
secondary antibody
Experiment 4
Run two different SDS-PAGE gels:
1. detect proteins by rapid Coomassie staining
2. detect protein by Western blot
Questions