Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3006: Procedure for Western Immunoblotting rat IGF-I

Introduction

The following protocol is used for the detection of rat IGF-I on a Western blot using Novozymes GroPep's rat IGF-I antiserum (rabbit).

Materials:

Either Tris buffered saline (TBS) or Phosphate buffered saline (PBS) can be used in this procedure.
For a 10 x concentrated TBS (Tris buffered saline) :
83 ml of 1.5M Tris (181.7 g/l)
42 g NaCl
Dilute to ~400 ml, pH to 7.6-8.0 and make up to 500 ml.

Tris buffered saline plus Tween 20 (TBS-T):
Dilute 10 x TBS 1/10 and add 0.05 % Tween 20

TBS - 3 % BSA (RIA Grade) - Blocking Reagent:
Bovine serum albumin (BSA) (RIA Grade) (3 g) dissolved gently with stirring in 90 ml sterile distilled or Milli-Q water.
Add 10 ml 10 x TBS
Filter through 0.22 µm Millipore filter

Primary Antibody:
Novozymes GroPep rat IGF-I antiserum (rabbit) (Product Code: PAT1)


GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech) ECL Western blotting Kit (RPN2108) contains:
ECL Reagents 1 and 2
Anti-rabbit IgG HRP conjugated whole antibody and
Anti-mouse IgG HRP conjugated whole antibody


Method:

1) Resolve sample by SDS-Page and transfer to nitrocellulose membrane using an electrotransfer unit.

2) Following transfer, rinse the nitrocellulose membrane with water. If desired the membrane can be stained with PonceauS reagent and destained with TBS (2 x 5 min).

3) Incubate membrane in 1 x TBS, 3 % BSA for 1-2 hour(s) at room temperature (or 4°C overnight).

4) Remove blocking solution and wash (4 x 8 min) with TBS - 0.05 % Tween 20.

5) Incubate with primary antibody (usually at 1/1000 or 1/2000 dilution in TBS-T) for at least 2 hours at room temperature (or 4°C overnight). Use 10 ml of antibody solution and rock gently.

6) Wash 4 x 8 min with TBS-T.

7) Incubate with anti-rabbit IgG, HRP-conjugated whole antibody (GE Healthcare Kit (formerly Amersham Biosciences / Pharmacia Biotech)) diluted 1/10,000 in TBS-T for 30 min at room temperature (volume 20 ml).

8) Wash 4 x 8 min with TBS-T.

9) Develop with ECL reagents at room temperature :
Using the GE Healthcare Kit (formerly Amersham Biosciences / Pharmacia Biotech) (RPN2108), mix 5 ml of reagent 1 with 5 ml of reagent 2, pour onto membrane, agitate 1 min, lightly blot with Whatman Filter Paper, wrap in Saran- / Glad-wrap and expose to film in cassette (10-30 sec depending on antibody). Develop film in dark room.


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