Professional Documents
Culture Documents
Load Proteins on
Gel
-
Apply Electric ---
Current
--
+ Proteins Separate by
--- ---
Transfer or Blot
-- --
Protein from Gel to
Nitrocellulose and/or
PVDF Membrane
Block
Membrane with
Non-Specific
Proteins
-- --
1o Antibody is a Rabbit
Anti-Human b-Actin
1o Antibody Binds Antigen Non-Specific Proteins
Antibody
(i.e. Protein of Interest) Bind to Unbound Regions
of Membrane
--- ---
Add HRP-Conjugated
2o Antibody
-- --
2o Antibody is a Goat
Anti-Rabbit-HRP-
Conjugated Antibody
Add
Chemiluminescen
t Substrate Luminol Ligh
t
Detected by
Film
HR
P
- -
Steps involved in western blotting
1. Sample preparation
2. Gel Electrophoresis
3. Blotting (or transfer)
4. Blocking
5. Antibody Probing
6. Detection
Sample Prep
Separate Proteins
Transfer proteins to
membrane
Block membrane
1ᵒ antibody
Wash
2ᵒ antibody
Wash
Detection
1- Sample Preparation
• All sources of protein, from single cells to whole
tissues, biological fluids and proteins secreted in
vitro, are open to analysis by Western blotting
• In most cases, the cells are harvested, washed,
and lysed to release the target protein
• For best results, all these steps should be carried
out on ice
• This will minimize proteolysis, dephosphorylation,
and denaturation, since all begin to occur once
the cells are disrupted
1- Sample Preparation
• All sources of protein, from single cells to whole
tissues, biological fluids and proteins secreted in
vitro, are open to analysis by Western blotting
• In most cases, the cells are harvested, washed,
and lysed to release the target protein
• For best results, all these steps should be carried
out on ice
• This will minimize proteolysis, dephosphorylation,
and denaturation, since all begin to occur once
the cells are disrupted
1- Sample Preparation
• The choice of extraction method depends
primarily on the sample and whether the analysis
is targeting all the proteins in a cell or only a
component from a particular subcellular fraction
• The endogenous proteases may be liberated
upon cell disruption and may degrade the target
molecule,
• the sample should be protected during cell disruption
and subsequent purification by the use of a cocktail of
protease inhibitors to avoid uncontrolled protein losses
1- Sample Preparation
• Numerous methods are available for disrupting cells and
preparing their contents for analysis by Western blotting
Detergent lysis The membranes are solubilized, lysing cells and
liberating their contents
Ultrasonication The sound waves generate a region of low pressure,
causing disruption of the membranes of cells
Freeze/thaw lysis Cells are disrupted by the repeated formation of
ice crystals and the method is usually combined with
enzymatic lysis
Enzymatic The enzymes dissolve cell walls, coats, capsules,
digestion capsids, or other structures