Western Blotting

1. Sample seperation
1. Homologous tissue crushed in buffer
  1. Centrifuged and supernatant removed
    1. Equal amount of SDS-buffer added
      1. Heated to denature the proteins
  2. Whole homogenate tested with equal amounts of SDS-buffer added
    1. Heated to denature the proteins
2. SDS-PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis)
1. electrical current is passed down an acrylamide gel pulling the proteins in order of size that is compared to a ladder
  1. the smaller the protein the further it can travel
2. Concentration of acrylamide
  1. the lower the acrylamide concentration the better the resolution of higher molecular weight proteins
  2. the higher the acrylamide concentration the better the resolution of lower molecular weight proteins.
3. measured in kilodaltons
4. 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
1. 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
2. Proteins are exposed on the surface of the membrane and are bound by hydrophobic and charged interactions
4. Probe with antibodies
1. 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
2. Step 2: Primary antibody is added to the target protein
3. Step 4: Secondary antibody (anti-antibody) with fluorophore is added to the primary antibody
4. Step 3: Excess primary antibodies washed off
5. Step 5: Excess Secondary antibody washed off
Detection
1. ECL reacts with the secondary antibody and transmits a signal that is proportional to the amount of antibody bound
Expose to Film
1. After incubation, the excess ECL reagent is drained off and the membrane is wrapped in cling film and taped into a light-tight cassette
2. 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
1. Develop film using suitable developing reagents
2. The longer the development the more the background signals come through
Densitometry and Analysis
1. Densitometry is the quantitative measurement of optical density in light-sensitive materials, such as film, due to exposure to light

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Western Blotting

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	Created by Ibi Qemlas over 8 years ago