Mirco Lab Midterm: Lecture 8, WESTERN BLOT Flashcards
What is Western Blot
- Powerful method to detect specific proteins or their
modifications in complex samples after separation by
electrophoresis and transfer to a solid support - Samples include whole cell lysates, tissue samples,
biological fluids, whole organisms, purified or partially
purified proteins - more popular in recent years has become the so-called
“Quantitative Western”, which upgraded the method from
qualitative (detection only or presence/absence) to
quantitative - assessing and comparing the amount of
target protein(s) in the sample or multiple samples
What is the Western Protocol?
Sample preparation, quantification
- Electrophoresis
- Transfer to a blotting membrane
- Blocking
- Incubation with primary antibody, wash
- Incubation with secondary antibody, wash
- Detection, imaging, and analysis
- IMPORTANT: Reliable western blotting results
demand optimization of each step
Describe the preparation
The goal must be to disrupt the cells/tissues to
release all proteins preferably in soluble form
without altering the protein of interest by
degradation, modifications, precipitation
- Always extract the proteins as quickly as
possible, on ice, in buffer with appropriate pH,
ionic strength, in the presence of protease
inhibitors
Protein extraction methods
Lysis with detergent: non-ionic (TRITON X-100, NP 40), RIPA
buffer (mix of ionic and non ionic detergents), direct lysis in SDS
electrophoresis sample buffer
- Mechanical disruption: Dounce, Potter-Elvehjem homogenizers,
blender, manual grinding with mortar and pestle, grinding with
abrasives (Glass beads, sand), ultrasonication - Enzymatic digestion (bacteria, fungi, plant, yeast)
- High pressure – French press
- Osmotic shock lysis , freeze/thaw lysis
- The extraction method depends on the source and is always a
combination of two or more of the above treatments
Sample cleaning
- No simple extraction protocol is perfect: for example
although not so crucial for 1D protein electrophoresis, a
cleanup of the samples after extraction might be necessary
for 2D protein electrophoresis - Cleanup/separation of cellular debris/insoluble materials by
centrifugation or filtration (warning: a protein of interest
might partition into soluble and/or insoluble fractions) - Nucleic acids: enzymatic digestion, ultrasonication
- Cleanup of salts, lipids, phenolic compounds (plants),
polysaccharides, ionic detergents most frequently by
precipitation with TCA and/or acetone or by dialysis
Total protein concentration in samples
- Comparing the amount of protein of interest by Western
blotting in samples run in parallel on the same gel or on
different gels requires that all lanes are loaded with known
total amount of protein - Total protein concentration can be determined using
standard curves, by spectrophotometric methods: UV,
Lowry, BCA, Bradford - IMPORTANT: Use commercial standards and kits
- IMPORTANT: Adjust the protein concentration with loading
buffer in order to load equal volume of samples on the gel
What is Electrophoresis
Western blotting requires the separation of
proteins by molecular weight and high
resolution
- Practically, it always employs one of the
denaturing (by SDS and reducing agents),
discontinuing gel electrophoresis systems
(Laemli – Tris glycine, Bis Tris MES/MOPS, Tris
tricine, Tris acetate)
Electrophoretic transfer (blotting)
Transferring the proteins after electrophoresis on solid
support membrane: multiple types of nitrocellulose or
PVDF (polyvinylidene difluoride)
- Proteins are transferred and fixed on the membrane at
their gel migrating positions and detected by
antibodies - Proteins are moved out of the gel toward the
membrane by electrical force, so during the blotting
they have to retain at least some charge (usually
negative due to remaining SDS
What Membranes are used?
Porous materials with pore sizes used in Western blots
between 0.1 and 0.45 μM
- Proteins bind to the membranes by non-covalent bonds
and hydrophobic interactions - Western blot sensitivity depends on the amount of protein
on the membrane and its accessibility to the antibodies - Two types of membranes are used: nitrocellulose and
PVDF. IMPORTANT: never use nylon membranes (the ones
used for nucleic acids binding) for Western – your blot
might end up with very high background
What are the Membrane characteristics?
Nitrocellulose: binds between 80-150 μg/cm2
protein, deficient
background can be used for any staining/detection,
methanol in transfer buffer improves protein binding,
incompatible with SDS in the transfer buffer, not recommended for
multiple stripping and re-probing with antibodies
- PVDF: binds 150-300 μg/cm2
protein binds small proteins
better than NC, excellent for stripping/re-probing, and allows
sequencing of bound proteins, not so sensitive to the presence
of small amounts of SDS in the transfer buffer, need to be prewetted in methanol before transfer in aqueous solution, tend
to have higher background than NC
What is Wet transfer?
The gel and membrane are fully immersed in transfer
buffer. Current is applied across the gel toward the
membrane. Requires cooling.
- Transfer is relatively slow, but more efficient and with
higher quality than semidry transfer. Transfers better large
proteins. - The gel is equilibrated in transfer buffer and assembled in
a sandwich: sponge-filter paper-membrane-gel-filter
paper-sponge. The membrane lies between the gel and
the anode (+). Membrane must be in a close contact with
the gel.
Semidry transfer
Uses less buffer, faster than wet transfer. The membrane
is in direct contact with the gel and several layers of filter
paper soaked in transfer buffer below the membrane and
above the gel. The sandwich is placed between the
electrode plates. Proteins move toward the membrane
and anode (+).
- Less efficient than wet transfer especially for large proteins
- Requires careful adjustment of the transfer conditions –
voltage and time to prevent overheating, drying or loss of
small proteins due to the high voltage gradient
Transfer buffer
There are many variations of transfer buffer depending on: wet or
semidry transfer, the membrane used, the molecular weight of
proteins to be preferably transferred
- Most transfer buffers contain methanol – strips SDS from the proteins
to ensure efficient binding of proteins, especially to NC membranes;
however, it shrinks the pores of the gel preventing the efficient transfer of
large proteins - SDS – excess SDS must be removed from gels by equilibration in
transfer buffer (10-30 min); decreases binding of proteins to
membranes, in particular NC; not so much for PVDF; however
complete stripping of proteins from SDS might prevent their transfer
to the membrane, especially large proteins, requiring low
concentrations 0.01-0.05% of SDS in the transfer buffer
Transfer buffer formulation
- Towbin buffer: 25mM Tris, 192mM glycine, 10-20% methanol, pH 8.3;
used for wet and semidry transfer; some variants contain 0.01-0.04% SDS - Bjerrum and Schafer-Nielsen buffer: 48mM Tris, 39mM glycine, pH
9.2,10-20% methanol; used for semidry transfer - CAPS buffer: 10mM CAPS, pH 11, 10% methanol; used for large proteins
and downstream sequencing - Discontinuous Tris-CAPS (for semidry only): 60mM Tris, 40mM CAPS pH
9.6, (+ 15% methanol on anode side and 0.1% SDS on cathode side);
provides efficient transfer - Dunn carbonate buffer: 10mM NaHCO3
, 3mM Na2CO3
pH9.9, 20%
methanol; may produce higher efficiency transfer and improve
antibodies binding
Monitoring transfer efficiency
Transfer efficiency varies among the proteins
- A common technique to check the quality of the
transfer (even transfer of proteins with different
MW from all lanes and parts of the gel, areas on
the membrane with no transfer – spots, smears)
is total protein staining on the membrane - IMPORTANT: It is recommended that total
protein staining and quantitation is used for
normalizing the quantitative Western blots
Total protein staining
Anionic dyes: Ponceau S, Coomassie, amido black, fast
green: low sensitivity (100-1000ng), inexpensive,
imaged by photography
- Fluorescence: SYPRO Ruby, sensitive (2-8ng), requires
imaging equipment (UV, LED epi-illumination, Laser
scanner) - Colloidal gold: very sensitive (100pg-1ng), expensive,
imaged by photography - Stain free: sensitive (2-20ng), special gels and
equipment required
What is blocking?
Blocking the sites on the membrane that can bind
proteins in order to prevent non-specific binding of
antibodies and high background signal
- In some cases blocking reagents can cause decrease of
the specific signal by competing with antibodies for
specific epitopes - Preliminary testing of a range of blocking
reagents/conditions is required to ensure low
background and no loss of signal in experiments with
new antibodies/antigen pairs or blotting protocol
What are Blocking reagents?
High concentration of proteins in buffers with or without detergents
(0.05-0.5% tween 20)
- BSA: 1-5%, NC and PVDF, can contain bovine IgG causing high
background with some secondary Ab - Non-fat dry milk, BLOTO, casein: 1-5%, NC and PVDF, do not use for
detection of phosphoproteins, can contain bovine IgG; can contain
endogenous Biotin that will interfere with biotin-streptavidin detection - Normal serum (serum from non-immunized host): reduces background
from non-specific conserved sequence binding; for best results must be
from the same host as the labeled secondary antibodies - Commercial blocking reagents with proprietary formula
what are Primary and secondary antibodies?
Primary antibodies bind specifically to the target protein
on the membrane. They are raised in host animals
injected with target protein as an antigen
- Secondary antibodies bind to the primary antibodies.
They are raised in animals injected with purified total (or a
specific subclass) immunoglobulins from the host animal
for the primary antibodies (rabbit anti goat antibodies are
raised in rabbits against total purified immunoglobulin
proteins from goat). Secondary antibodies are usually
conjugated with enzymes or labeled with fluorescent dye
used in immunodetection.
What are Polyclonal and monoclonal primary
antibodies
Antibodies recognize short stretches of amino acids
(epitopes) of their protein targets
- Polyclonal antibodies recognize multiple epitopes on a
single protein (antigen) and usually have higher
affinity, lower specificity - Monoclonal antibodies recognize single epitope from
the antigen and have lower affinity but higher
specificity - Both require affinity purification before use for
Western blotting
Secondary antibodies
Practically always commercial
- Affinity purified with ProteinA/G to separate IgGs from
other plasma proteins or with IgGs (from the host of
primary antibodies) to select for secondary antibodies that
bind specifically only to primary antibodies. In addition
they can be cross-absorbed with immunoglobulins from
species that are evolutionary close to the primary
antibody host to achieve minimal cross-reaction with them
(i.e. when multiplexing). - Commercially conjugated to enzymes (AP or HRP mostly),
fluorescent dyes, biotin
Antibody validation – important terminology
Ab specificity: ability to bind the intended target
- Ab selectivity: degree of preference for the target protein in the
presence of other sample proteins - Ab affinity: strength of binding to an epitope
- Cross-reactivity: non-specific binding to other sample proteins
with epitopes similar to the target protein. IMPORTANT:
selectivity and cross-reactivity depend on relative abundance of
the target protein. For example different target protein
concentrations in the sample and antibody concentrations used
for detection can lead to different levels of off-target (nonspecific) Ab binding even if the Ab affinity to non-specific targets
is much lower than the affinity to the target.
What are Antibodies validation for Western?
Demonstrate Ab specificity and selectivity for
endogenous levels of target protein – not for purified
protein - using Ab from different sources and different
Ab dilutions (more sensitive assays require higher Ab
dilutions: primary 1:500-1:5000, secondary 1:5000 –
1: 100000)
- Examine antibody binding to cell lines/tissues with
different levels of target protein expression - Whenever possible include positive and negative
(samples not expressing target protein) controls
Choice of labeled Antibodies
- Enzymes: alkaline phosphatase and horseradish
peroxidase - The substrates for the detection with both enzymes
are chromogenic, chemiluminescent or
chemifluorescent - Fluorescent dyes
- Biotin
- IMPORTANT: labeled primary Ab are sometimes used
in Western blotting to improve specificity, but
detection with them is less sensitive
Imaging with visible light scanners
Densitometers: these are in essence scanners, which
use visible light for analysis of gels and X-Ray film after
chemiluminescence detection (in transmission mode),
blots (in reflection mode) stained with visible light dyes
- Excellent for documentation, however if intending to do
quantitative analysis of images use scientific scanners
(rather expensive). Office scanners have several
problems: narrow dynamic range, have an automatic
gain control (OD at certain location of the scan depends
on the surrounding areas), uneven illumination
Imaging with CCD cameras
- Versatile systems for gels and blots. Can be used with
trans-illumination by light boxes (Visible and UV) for gels
and X-ray films or epi-illumination of blots after
chromogenic or fluorescence detection. - Supercooled CCD cameras (Expensive!) are used without
illumination to directly capture chemiluminescence of
western blots without first documenting it on X-ray film.
The advantage is direct digitalization, broader dynamic
range (2-5 orders of magnitude), improved sensitivity
necessary for quantitation
Imaging with laser scanners
Offers the highest sensitivity, resolution, and linear dynamic
range (up to 104
-105
orders of magnitude), uniformity
across the entire scan area for gels and blots stained
with fluorescent dyes and stain-free technology
- Laser source provides an excitation beam that is highly
monochromatic, which contributes to the improved
sensitivity and resolution - Allows multiplexing: detection of multiple fluorescent
dyes on the same blot (gel) - Excellent for quantification of Western blots
