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
