Dot Blot assay for Bacillus pumilus nitrilase

Dot blot is a technique for detecting, analyzing, and identifying proteins, similar to the Western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane.

Dot blot technique is being used in our laboratory for the detection of Bacilluspumilus Nitrilase protein. For primary and secondary (conjugated with alkaline phosphatase) antibodies specifics plus their working concentrations, visit this site soon.

Equipment and apparatus

Hybond TM -C Nitrocellulose hybridization membranes

Reagents

Tris-EDTA-Nacl buffer (TEN) [25 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6]. 3.03 g Tris, 0.29 g EDTA, and 8.77 g NaCl are dissolved in dd.H₂O. The solution is titrated to pH 7.6 using HCL, and then made up to 1 l with dd. H₂O.

Blocking solution [3% BSA in TEN]; 3 g BSA are dissolved in 100ml TEN

Wash Buffer [0.1% Tween20 in TEN buffer]. 0.1ml Tween20 are mixed with 100 ml TEN buffer.

Alkaline phosphatase detection buffer [50 mM Tris-HCl, 5 mM MgCl₂; pH 9.5]. 0.61 g Tris and 0.1 g MgCl₂ are dissolved in water and made up to 100 ml.

Alkaline phosphatase substrate solution [0.015% (w/v) BCIP, 0.03% (w/v) NBT in detection buffer]. BCIP (1.5 mg in 1 ml DMF), NBT (3 mg in 1 ml alkaline phosphatase detection buffer) and 10 ml detection buffer are mixed well just before use. BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate) and NBT (P-Nitro Blue Tetrazolium chloride) stocks are stored at -20°C and 4°C, respectively.

Antibodies stocks : These are made to final working concentrations before use.  

Procedure

1. Nitrocellulose membrane is labeled appropriately (with a pencil) for protein elution fractions.
2. 2 μl are pipetted from each fraction onto the membrane, and the membrane allowed to dry.
3. When dry, the membrane is incubated in blocking solution for 1 hour.
4. After incubation, the membrane is immersed into the primary antibody solution (diluted in blocking solution), followed by 2 hours shaking at room temperature.
5. The membrane is washed in buffer (3*10 min), after which it is transferred into secondary antibody-alkaline phosphatase enzyme conjugate solution (in blocking solution) for 1 hour.
6. The membrane is washed in buffer as before (3*10 min), after which it is transferred to substrate solution, until spots are visible.
7. The reaction is stopped, substrate solution poured out and the membrane rinsed in distilled water
8. The membrane is air-dried