Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3110: Protocol for Western Immunoblotting using human IGFBP antisera

Introduction

The following protocol is used for the detection of IGFBPs on a Western blot using Novozymes GroPep's human IGFBP antisera.

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)
42g NaCl
Dilute to ~400 ml, pH to 7.6-8.0 and make up to 500ml.

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

TBS - 3%BSA - Blocking Reagent
Bovine serum albumin (BSA) (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 human IGFBP antisera

Catalog Codes:

Anti-human IGFBP-1 (PAP1)
Anti-human IGFBP-2 (PAQ1)
Anti-human IGFBP-3 (PAV1)
Anti-human IGFBP-4 (PAR1)
Anti-human IGFBP-5 (PAZ1)
Anti-human IGFBP-6 (PAU1)


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 distilled or Milli-Q 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 4C 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 4oC overnight). Use 10 ml of antibody solution and rock gently.

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

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

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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