Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3009: Procedure for Western Immunoblotting human ProIGF-II

Materials:

Use Tris buffered saline (TBS) for 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 0.1 % (v/v) Tween 20

Blocking Reagent: TBS - 5 % Non-Fat Milk Powder
Non-fat milk powder (5 g) dissolved gently with stirring in 90 ml sterile distilled or Milli-Q water.
Add 10 ml 10 x TBS. Add 0.1% (v/v) Tween 20.
Filter through 0.22 µm Millipore filter

Primary Antibody:
Novozymes GroPep Pro-IGF-II antisera (100 µl)

Anti-human ProIGF-II (aa 78-88) (Product Code: PAAZ1)
Anti-human ProIGF-II (aa 89-101) (Product Code: PAAY1)
Anti-human ProIGF-II (aa 138-156) (Product Code: PAAX1)

GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech) ECL Western blotting Kit containing:
ECL Reagents 1 and 2
Anti-rabbit Ig 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 with 5 % non-fat milk powder in TBS + 0.1 % Tween 20 (TBS-T) for 1-2 hours at room temperature.

4) Remove blocking solution and wash 2 x 8 min TBS-T.

5) Incubate overnight at 4°C with primary IGF-IIE antibody at 1/1,500 in 1 % non-fat milk powder in TBS-T. 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 (GE Healthcare Kit (formerly Amersham Biosciences / Pharmacia Biotech)) diluted 1/5,000 in TBS-T for 60 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), 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.


© Novozymes GroPep Limited
28 Dalgleish Street
Thebarton SA 5031
PO Box 10065 BC
Adelaide SA AUSTRALIA
Telephone +61 (0)8 8354 7700
Facsimile +61 (0)8 8354 7788
Email: AUAD-bioreagents@novozymes.com
Website: www.gropep.com.au