protein detection methods & western blots
TRANSCRIPT
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
•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
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SDS-PAGE: The Matrix
Free radicals
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
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SDS-PAGE: The buffer system
Stacking gel: 4%
Discontinuous Laemmli buffer system - buffer
in the gel and the tank are different
Resolving gel: range 5-20%
Gly
proteinsCl-
Proteins are concentrated into the narrow
zone between the Cl- and glycine fronts.
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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 stainingStain Advantages Disadvantages
CoomassieBlue
Simple methodologyMS compatibleEasy to image
Very insensitive
Bio-SafeColloidalCoomassieStain
Simple methodologyMS compatibleMore sensitiveEasy to image
Less sensitive thansilver
Silver stain Very sensitiveEasy to image
Complex procedureIncompatible with MSNot quantitative
Sliver stainPlus
Very sensitiveEasy to imageMore MS compatible
than standard silverstaining
Complex procedureNot very MS
compatibleNot quantitative
Ruby stain Very sensitiveMS compatibleQuantitative over 3
orders of magnitudeSimple methodology
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)
Comparative Proteomics Kit II:
Western Blot Module
Bio-Rad’s take on Western blotting!
Why Teach
Western Blotting?
• Powerful teaching tool
• Real-world connections (?)
• Laboratory extensions (?)
• Tangible results
• Link to careers and industry (?)
• Standards-based (?)
According to Bio-Rad
Why use Western blotting?
•Can specifically determine if your protein of
interest is present in a crude protein preparation
Transfer
Proteins from
the gel to the
nitrocellulose
membrane
60 minutes
200 mA
Blotting buffer
1x Tris glycine
with 20%
methanol,
0.1% SDS
Electric Current
Protein Transfer
Blocking
Buffer
Remove membrane from the blotting sandwich
and immerse in blocking solution
5% non-fat milk or 1% gelatin: Prevents the
primary antibody from binding randomly 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
Blocking membrane before addition of antibody
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 1o 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 2o 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 2o 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 the enzyme substrate (HRP color detection reagent) to
the membrane
•Incubate for 10 minutes
•The colorimetric substrate is cleaved by the enzyme
conjugated (attached) to the secondary antibody
Other detection methods
•Chemiluminescent substrates
• Film or cooled CCD camera
•Infrared emitting side groups
• Special machine
•Quantum dots
• Epi UV and CCD camera with EtBR filter
Western vs ELISAEnzyme-Linked Immunosorbant Assay
ELISA
- Quick results
- Primary screening
- Identifies proteins by
antibody specificity only
- With correct controls
can be quantitative
Western Blot
- Confirm ELISA results
- More specific
- Identifies proteins by both antibody specificity and size
-Difficult to use for quantification
Questions ?