1) Eelectrophoretic separation of protein or of nucleic
acid fragments in the sample
2) Transfer to and immobilization on ...
Western blot
Protein Detection
• Detects proteins and estimates their molecular
weight.
• Detects changes in phosphorylation and lipid
modifications.
• U...
Sample Preparation
SDS-PAGE (Protein Electrophoresis)
Electro-transferring
Immunoblotting
• Major components of the sample loading “buffer”
– SDS
– DTT
– Tracking dye
– Glycerol
– Protease inhibitor
Western Blot Visual Protocol: Phase 1: Sample Preparation
• Sodium dodecyl sulfate (SDS)
• Tris buffer (either glycine or tricine)
• Acrylamide and NN-bis-acrylamide
– Forms gel ma...
Stacking (concentrating) gel
4% acrylamide
0.5M Tris-H+Cl-, pH 6.8, 0.1%
SDS
Resolving (separating) gel
10% acrylamide (36...
How to Make an SDS-PAGE gel
Western Blot Visual Protocol: Phase 2: Protein Electrophoresis
• uses an electric current to pull proteins from the gel
into the PVDF or nitrocellulose membrane
• Transfer buffer contai...
• Equilibrate gel in transfer buffer
in separate tray.
• Equilibrate filters and sponges
in transfer buffer.
• PVDF membra...
Western Blot Visual Protocol: Phase 3: Membrane Transfer
• Washing (TBS-T)
• Blocking
• Incubation with Primary antibody
• Washing (TBS-T)
• Incubation with secondary antibody
• Blocking reduces
nonspesific binding of
antibody (primary or
secondary) to protein or
membrane
• Too little => high
back...
• After blocking membrane, add antibodies in to
blocking solution (ie 5% milk).
• Incubate overnight at 4oC or 2 hours at ...
• Buffer: PBS w/Tween 20 or TBS w/Tween 20
– TW20 concentration must be determined for each antibody and
antigen
– Usually...
• Buffer: same as for primary antibody
• Dilution must be determined in each case
– Usually 1:1,000 - 1:100,000
• Incubati...
• Colorimetric detection
• Chemiluminescent detection
• Radioactive detection
• Fluorescent detection
Western Blot Visual Protocol: Phase 4: Immunoblotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
Protein Immunoblotting- An Introduction  to Western Blotting
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Protein Immunoblotting- An Introduction to Western Blotting

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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.

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Transcript of "Protein Immunoblotting- An Introduction to Western Blotting"

  1. 1. 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
  2. 2. Western blot Protein Detection
  3. 3. • Detects proteins and estimates their molecular weight. • Detects changes in phosphorylation and lipid modifications. • Used to detect changes in protein expression.
  4. 4. Sample Preparation SDS-PAGE (Protein Electrophoresis) Electro-transferring Immunoblotting
  5. 5. • Major components of the sample loading “buffer” – SDS – DTT – Tracking dye – Glycerol – Protease inhibitor
  6. 6. Western Blot Visual Protocol: Phase 1: Sample Preparation
  7. 7. • 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
  8. 8. 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
  9. 9. How to Make an SDS-PAGE gel
  10. 10. Western Blot Visual Protocol: Phase 2: Protein Electrophoresis
  11. 11. • 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
  12. 12. • 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
  13. 13. Western Blot Visual Protocol: Phase 3: Membrane Transfer
  14. 14. • Washing (TBS-T) • Blocking • Incubation with Primary antibody • Washing (TBS-T) • Incubation with secondary antibody
  15. 15. • 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
  16. 16. • After blocking membrane, add antibodies in to blocking solution (ie 5% milk). • Incubate overnight at 4oC or 2 hours at room temperature.
  17. 17. • 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
  18. 18. • 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
  19. 19. • Colorimetric detection • Chemiluminescent detection • Radioactive detection • Fluorescent detection
  20. 20. Western Blot Visual Protocol: Phase 4: Immunoblotting
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