Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3520: Procedure for Western Immunoblotting of EGF / Betacellulin

Introduction

The following protocol is used for the detection of human Epidermal Growth Factor (EGF) on a Western blot using Novozymes GroPep’s human EGF polyclonal antiserum (Catalog Code PAD1) and the ECL Western blot detection system supplied by GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech). This protocol is more sensitive than the traditional colorimetric methods. For EGF Western blotting, we suggest that the EGF should be blotted onto 0.22 µm nitrocellulose membrane.


Materials
1. Tris-buffered saline (TBS): 150 mM NaCl, 10 mM Tris base, pH 7.5.
Dissolve 2.42 g Tris base and 17.5g NaCl in 1.5 litres distilled or Milli-Q water, adjust pH to 7.5 using concentrated hydrochloric acid and make up to a final volume of 2 litre with distilled or Milli-Q water.

2. 3 % BSA/TBS: Dissolve 3 g bovine serum albumin (BSA) (Sigma, Catalog #A7888) in 100 ml TBS.

3. 0.5 % BSA/TBS: Prepare a TBS solution containing 0.5 % BSA from the 3 % BSA/TBS stock and TBS.

4. Novozymes GroPep’s anti-human EGF polyclonal antiserum (Catalog Code PAD1).

5. 0.1 % Tween 20/TBS: Mix 0.5 ml Tween 20 (Sigma, Catalog #P1379) thoroughly with 500 ml TBS.

6. Secondary antibody: Peroxidase-conjugated swine anti-rabbit immunoglobulins (Dako Corporation, CA, USA, Catalog #P448).

7. ECL Western blot detection system (GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech), Catalog #RPN 2106).


Method

All steps should be carried out on a shaker

1. Block non-specific binding sites by incubating the membrane in 3% (w/v) BSA/TBS for 1 hour at room temperature or overnight at 4°C.

2. Wash membrane twice with at least 50 ml of TBS, 10 minutes each.

3. Incubate the membrane for 1 hour with Novozymes GroPep’s anti-human EGF polyclonal antiserum (Catalog Code PAD1) at a final dilution between 1/1,000 to 1/5,000 in 0.5% (w/v) BSA/TBS.

4. Rinse the membrane briefly with 2 changes of TBS, then wash it 3 times in 0.1% Tween 20/TBS, each time for 15 minutes.

5. Incubate membrane for 1 hour with the secondary antibody diluted at 1/5,000 in 0.5 % BSA/TBS.

6. Wash as for step (4).

7. Develop the blot with GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech) ECL Western Detection System following the supplier's instructions.


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