Zusammenfassung der Ressource
Western Blotting
- 1. Sample seperation
- Homologous tissue crushed in buffer
- Centrifuged and supernatant removed
- Equal amount of SDS-buffer added
- Heated to denature the proteins
- Whole homogenate tested with equal
amounts of SDS-buffer added
- Heated to denature the proteins
- 2. SDS-PAGE (Sodium Dodecyl Sulphate
PolyAcrylamide Gel Electrophoresis)
- electrical current is passed down an acrylamide gel pulling the
proteins in order of size that is compared to a ladder
- the smaller the protein the further it can travel
- Concentration of acrylamide
- the lower the acrylamide concentration the better the resolution of higher molecular weight proteins
- the higher the acrylamide concentration the better the resolution of lower molecular weight
proteins.
- measured in kilodaltons
- The stacking gel is more acidic than the resolving gel. This concentrates the proteins in to a tight
stack. As the pH increases whilst the proteins move through the resolving gel, the proteins are able to
resolve from one another and move according to their size.
- 3. Transfer to membrane
- SDS-PAGE and membrane is sandwiched together, this allows another metrical current to be
passed. This 'pulls' the proteins to the exact position on the membrane
- Proteins are exposed on the surface of the membrane and are bound by
hydrophobic and charged interactions
- 4. Probe with antibodies
- Step 1: Blocking = prevent the detecting antibodies binding
non-specifically to the surface of the membrane, the membrane
surface is coated first with a dilute solution of protein
- Step 2: Primary antibody is added to the target protein
- Step 4: Secondary antibody (anti-antibody) with fluorophore
is added to the primary antibody
- Step 3: Excess primary antibodies washed off
- Step 5: Excess Secondary antibody washed off
- Detection
- ECL reacts with the secondary antibody and transmits a signal
that is proportional to the amount of antibody bound
- Expose to Film
- After incubation, the excess ECL reagent is drained off and the membrane is
wrapped in cling film and taped into a light-tight cassette
- In a darkroom, film is placed directly on top of the membrane to expose it
to the light produced from the ECL reaction
- Develop film
- Develop film using suitable developing reagents
- The longer the development the more the background signals come through
- Densitometry and Analysis
- Densitometry is the quantitative measurement of optical density in
light-sensitive materials, such as film, due to exposure to light