Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3003: Procedure for Western Ligand Blotting using iodinated IGF-I or IGF-II

Introduction

The following protocol is used for the detection of IGF Binding Proteins using Novozymes GroPep's iodinated IGF-I or IGF-II. The IGFBP sample is resolved by gel electrophoresis, followed by electrotransfer to Nitrocellulose and detection by X-Ray film or Phosphoimager after the binding of the iodinated IGF-I or IGF-II to the IGFBP(s).

Equipment:
Gel system (e.g. Novex XCell Sure Lock (Invitrogen) and Electrotransfer unit (e.g. LKB Pharmacia NovaBlot Multiphor II). Details are given for these units, but follow instructions for the particular apparatus being used.

Materials:
Gels, Nitrocellulose, Whatman paper (1M). GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech) ECL Reagents 1 and 2 (RPN2209).

Reagents:
Use Novozymes GroPep's iodinated IGF-I or IGF-II (human, chicken, fish or rat).

See Protocol:
#3001 Procedure for the Iodination of IGFs.

Tris base (BDH 103157P), Tween 20 (Sigma P7949), Bovine Serum Albumin (BSA) (Sigma A7888), Avidin-HRP (GE Healthcare (formerly Amersham Biosciences / Pharmacia Biotech) RPN1231).

Stains:
Coomassie Blue (Sigma B8522), Ponceau S reagent (Sigma P7170).

Solutions:

Anode (-) 1 buffer:
36.3 g Tris(hydroxymethyl)methylamine
200 ml Methanol
Make up to 1 litre with distilled or Milli-Q water.

Anode (-) 2 buffer:
3.03 g Tris(hydroxymethyl)methylamine
200 ml Methanol
Make up to 1 litre with distilled or Milli-Q water.

Cathode (+) buffer:
5.2g 6-Aminocaproic acid (6-Amino n-hexanoic acid)
200 ml Methanol
Make up to 1 litre with distilled or Milli-Q water.

Saline Solutions

Tris buffered saline (TBS) is used in this Western Ligand blotting procedure.

For a 10 x concentrated TBS (Tris buffered saline) :
83 ml of 1.5 M 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 1 % Tween 20 (TBS-1 % T)
Dilute 10 x TBS 1/10 and add 1 % (v/v) Tween 20

Tris buffered saline plus Tween 20 (TBS-0.1 % T)
Dilute 10 x TBS 1/10 and add 0.1 % (v/v) Tween 20

Tris buffered saline plus Tween 20 plus BSA (TBS-0.1 % T-1 % BSA)
0.1 % Tween 20/TBS + 1 % BSA (200 mg/20 ml TBS)


Method:

1) Choose the gel best suited to separate the M Wt range of the protein(s) being studied.

2) Run the loaded gel under non-reducing conditions, using the standard loading buffer that is recommended for the gel. The gel can be stained with Coomassie Blue after the transfer to Nitrocellulose.


Electro-transfer of the protein to nitrocellulose using a semi-dry transfer apparatus:

3) Wearing latex gloves to prevent contamination, 18 pieces of 9 x 6 cm Whatman 1mm filter paper are cut out for each gel. 6 pieces are soaked in Anode 1 buffer, 3 pieces in Anode 2 buffer, 9 pieces in Cathode buffer and 1 piece of nitrocellulose (9 x 6 cm) is soaked in Anode 2 buffer.

4) The paper, nitrocellulose and gel are then stacked onto the graphite electrotransfer plates, in the following order, starting from the bottom:-

6 (Anode 1) on graphite
3 (Anode 2)
Nitrocellulose
Gel
9 (Cathode) on top.

5) The transfer is run at 43 mA per nitrocellulose strip for at least 1 hour. In some cases 2 hours of transfer may be necessary to give better results.

6) Check that the protein has transferred satisfactorily to the Nitrocellulose by staining with Ponceau solution:

Place the Nitrocellulose in neat Ponceau solution for 1-2 mins, then destain with distilled or Milli-Q water. If the protein has transferred satisfactorily, bands will be visible. If bands are visible, the nitrocellulose can then be either washed immediately (see below) or stored dry, depending on the time schedule.

If the transfer of the protein to Nitrocellulose is not satisfactory, destain the Nitrocellulose completely with distilled or Milli-Q water and then continue with the electrotransfer.

Washing of the Nitrocellulose Membrane:

7) The Nitrocellulose is washed by shaking with about 20 ml of TBS-1 % T for 30 mins, room temperature. Discard wash solution.

8) The Nitrocellulose is then blocked with 20 ml TBS-0.1 % T-1 % BSA, preferably overnight at 4°C (or for 2 hours room temperature). Pour this blocking solution off when finished and save it for the radioactive ligand.

9) Wash the Nitrocellulose in TBS-0.1 % T for 10 minutes.

10) For probing with ¹²⁵I-IGF-I approximately 1,000,000 cpm of ligand is used per nitrocellulose strip. The tracer is added to the TBS-0.1 % T-1 % BSA blocking solution saved from Step (8) (20 ml is usually sufficient) and washed for 2 hours, room temperature (or overnight at 4°C).

11) Wash in TBS-0.1 % T for 30 mins.

12) Wash 5 x 5 mins in TBS-0.1 % T.

13) Dry the Nitrocellulose on Whatman paper and wrap in gladwrap to prevent contamination.

14) Place and tape the Nitrocellulose onto the inside of an autoradiography cassette. Place the X-ray film on top and leave at –80°C overnight (or longer if desired). Develop the X-ray film in the appropriate apparatus (3-5 min).

Alternatively, the Nitrocellulose can be analysed in a Phosphorimager machine.


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