Western blotting is an important technique used in cell and molecular biology. The specificity of the antibody-antigen interaction enables a target protein to be identified from a complex protein mixture. Western blotting can produce qualitative and semi-quantitative data about that protein. This powerpoint will attempt to explain the technique and theory behind western blot.

3. 1) Eelectrophoretic separation of protein or of nucleic
acid fragments in the sample
2) Transfer to and immobilization on a matrix.
3) The probe is added to the matrix to bind to the target
molecules.
4) Any unbound probes are then removed.
5) Visualization of bound probe

4. Western blot
Protein Detection

6. • Detects proteins and estimates their molecular
weight.
• Detects changes in phosphorylation and lipid
modifications.
• Used to detect changes in protein expression.

8. Sample Preparation
SDS-PAGE (Protein Electrophoresis)
Electro-transferring
Immunoblotting

9. • Major components of the sample loading “buffer”
– SDS
– DTT
– Tracking dye
– Glycerol
– Protease inhibitor

10. Western Blot Visual Protocol: Phase 1: Sample Preparation

11. • Sodium dodecyl sulfate (SDS)
• Tris buffer (either glycine or tricine)
• Acrylamide and NN-bis-acrylamide
– Forms gel matrix
• TEMED
– Catalyst for polymerization (produces free radials from APS)
• Ammonium persulfate (APS)
– Source of free radials for polymerization
Could purchase pre-cast gels if you have the
money.
Ingredients in Gel

12. Stacking (concentrating) gel
4% acrylamide
0.5M Tris-H+Cl-, pH 6.8, 0.1%
SDS
Resolving (separating) gel
10% acrylamide (36.5:1,
acryl/bis)
1.5 M Tris-H+Cl-, pH 8.8, 0.1%
SDS
Running buffer
0.25 M Tris base; 1.92 M
glycine, pH 8.3; 1% SDS
Resolving gel
Stacking gel
Discontinuous system

13. How to Make an SDS-PAGE gel

14. Western Blot Visual Protocol: Phase 2: Protein Electrophoresis

15. • uses an electric current to pull proteins from the gel
into the PVDF or nitrocellulose membrane
• Transfer buffer contains: Tris, Glycine, and methanol
but no ions.
• Nitrocellulose membranes are cheaper than PVDF,
but are far more fragile and do not stand up well to
repeated probings
• Western blot transfer can be done in wet or semi-
dry conditions

16. • Equilibrate gel in transfer buffer
in separate tray.
• Equilibrate filters and sponges
in transfer buffer.
• PVDF membranes must be
soaked in methanol, before
equilibration in transfer buffer.
Nitrocellulose membranes are
soaked directly in transfer
buffer
 Mount transfer sandwich in
blotting chamber which already
contains transfer buffer

17. Western Blot Visual Protocol: Phase 3: Membrane Transfer

20. • Washing (TBS-T)
• Blocking
• Incubation with Primary antibody
• Washing (TBS-T)
• Incubation with secondary antibody

21. • Blocking reduces
nonspesific binding of
antibody (primary or
secondary) to protein or
membrane
• Too little => high
background
• Too much reduces the
signal
• Incubation time:
– 1-2 hrs at RT with shaking
• Blocking agents
– Fat free dry milk
– Bovin serum albumin
– Casein
– Gelatin
– Hemoglobin
– Ovalbumin
• Buffer:
– PBS, phosphate buffered saline, pH
7.5-8.0
– TBS, TRIS-buffered saline, pH 7.5

22. • After blocking membrane, add antibodies in to
blocking solution (ie 5% milk).
• Incubate overnight at 4oC or 2 hours at room
temperature.

23. • Buffer: PBS w/Tween 20 or TBS w/Tween 20
– TW20 concentration must be determined for each antibody and
antigen
– Usually 0.01-0.2%
• Time: Number of washes and duration of each wash
must be determined in each case
– Usually 3X5 min
– Use large buffer volume: 50-100 ml for 8X10 cm membrane
– Incubation with vigorous shaking

24. • Buffer: same as for primary antibody
• Dilution must be determined in each case
– Usually 1:1,000 - 1:100,000
• Incubation time must be determined in each case
– Varies from 5 min to 2 hrs
enzym

25. • Colorimetric detection
• Chemiluminescent detection
• Radioactive detection
• Fluorescent detection

26. Western Blot Visual Protocol: Phase 4: Immunoblotting