Dot Blot

Dot Blot (DB)

The size of nitrocellulose membrane used is dependent on the number of tests needed, these numbers are for reference only. As a guide, the primary antibody can be diluted using blocking buffer to a final concentration of 0.5 ?g/ml. Concentrations will depend on the sample used and should always be optimised by the end user.

A. Sample preparation

Reagents

RIPA Buffer (Radio Immuno Precipitation Assay Buffer) contains:

  • 50 mM Tris, pH 8.0
  • 150 mM NaCl
  • 1% NP-40
  • 0.5% Sodium Deoxycholate
  • 0.1% SDS (Sodium dodecyl sulphate)

Protease Inhibitors

  1. Remove media.
  2. Wash cells twice to remove residual media using PBS.
  3. Remove PBS and add appropriate volume of RIPA Buffer plus protease inhibitors (1 ml per 0.5 to 5 x 107 cells). Incubate at 4°C for 5 min.
  4. Dislodge cells using a cell scraper and transfer to a tube at 4°C. (NB. If needed, store at -70°C at this point. Storing after denaturation may cause aggregation)
  5. Spin the lysate at 8000 x g for 10 min at 4°C to pellet the cell debris. (NB. Before centrifugation, remove nucleic acid aggregates which may form at the top using a pipette.)
  6. Transfer supernatant containing the soluble protein to a new tube at 4°C.

B. Membrane preparation

  1. Cut the nitrocellulose membrane into a 2x5 cm rectangle. Draw a 1x1 cm grid on the membrane using pencil. Label the 10 resulting squares, for example 1-8 samples, 2 controls.
  2. Serially dilute samples using PBS (pH 7.4).
  3. Blot 2 µl of each sample into the centre of each grid.
  4. Allow to dry for 30 min.
  5. Incubate the membrane in blocking buffer (5% (w/v) non-fat milk in TBST) for 1 hour at room temperature.

C. Immunoblotting

  1. Incubate the membrane with primary antibody at appropriate concentration for ~1 hour at room temperature.
  2. Wash the membrane with TBST (pH 7.4) for 5 min on a shaker at room temperature. Repeat twice.
  3. Incubate the membrane with secondary antibody at appropriate concentration in blocking buffer and incubate for ~1 h on shaker at room temperature.
  4. Wash the membrane with TBST (pH 7.4) for 5 min on a shaker at room temperature. Repeat twice.

D. Detection

  1. Detection will depend on the conjugation of your primary or secondary antibody. For fluorescently labelled antibodies, use a fluorescence scanner, as per the manufacturer’s instructions. For HRP conjugated antibodies, use an enhanced chemiluminescence (ECL) kit and follow manufacturer’s guidelines.