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Western Blotting

THE RECOMMENDATIONS BELOW REPRESENT GENERAL GUIDELINES AND ARE NOT MEANT TO BE ABSOLUTES.  OPTIMIZATION OF ASSAY CONDITIONS IS THE RESPONSIBILITY OF THE RESEARCHER.

Basic buffers:

TBS: 8.76 grams of NaCl and 2.42 grams of Tris base in 900 ml of deionized water. Adjust pH to 7.4 with 6N HCl. Adjust the volume to 1 liter with deionized water.

PBS: 8.0 grams of NaCl, 0.2 grams of KCl, 1.44 grams of Na2HPO4 and 0.24 grams of KH2PO4 in 900 ml of deionized water. Adjust pH to 7.4. Adjust volume to 1 liter with deionized water.

Blocking buffer: TBS or PBS containing one of the following: 2%(w/v) BSA, ovalbumin or casein, or 5%(w/v) non-fat powdered milk.
BSA/Tween buffer: TBS or PBS containing 0.02%(w/v) BSA and 0.05% (v/v) Tween 20.

Blocking conditions: After the protein samples have been transferred from the gel to either a nitrocellulose or PVDF membrane, remaining binding sites are blocked by incubating the blot in blocking buffer for 2 hours at room temperature or overnight at 4oC. Be sure to gently mix or agitate the blot during all blocking and incubation steps.

Wash steps: Wash the blot at least three times (incubating for 3 minutes with each wash) with BSA/Tween buffer between each procedural step.

Primary antibody: Primary antibodies should be diluted in BSA/Tween buffer.  Dilute monoclonals and affinity purified polyclonals to 5 micrograms per ml. Dilute non-affinity purified polyclonals to 25 micrograms per ml. Incubate the blot with the primary antibody for 2 hours at room temperature, or overnight at 4oC.  These dilutions are also recommended if your primary antibody is conjugated.  Further optimization of these conditions may be necessary for your application.

Secondary antibody: Use a peroxidase conjugated secondary antibody that is appropriate for your primary antibody (i.e., if you are using a murine IgG monoclonal, you may select a peroxidase conjugated goat anti-mouse IgG). Dilute the secondary antibody according to the manufacturer’s suggestions. Incubate with the blot for 1 to 2 hours at room temperature.

Development: Development may be done using the DAB (3,3'-diaminobenzidine) detection system (see Sigma catalog #D4418) or by chemiluminescence (see Amersham ECL Western Blotting Detection Reagents). Follow the manufacturer’s instructions for either application.