Procedure for non-radioactive western-ligand blot for the detection of human bovine, porcine, rabbit, guinea pig, sheep, donkey and goat or mouse and rat IGFBP´s and IGF receptors using biotinylated IGF-II or biotinylated IGF-I:

Detects IGFBP's and IGF-Receptors in samples of human origin. Similar patterns are obtained with samples from bovine, porcine, rabbit, guinea pig, sheep, donkey and goat serum. Only weak bands are obtained with rat and mouse serum on nitrocellulose membranes (see below : Mouse/rat western-ligand blotting kit).

Some Examples of Western-Ligand Blots of IGFBP´swith Human IGF-I, IGF-II and Analogs can be seen in application note:

IGF004 Applications of Biotinylated IGF´s and analogs

Examples of Western-liagnd blots of receptors are found in Reference 5 and 6.

Contents of the human and other species western-ligand blot kit:

The reagents of the kit are sufficient for the preparation of 250 ml of each reagent in each step. This is sufficient for 500 square-cm of blotting membrane. Each reagent of the kit is also available separately.We routinely use biotinylated IGF-II as ligand! Alternatively biotinylated IGF-I may be used. However, IGF-I presumably binds only weakly to mouse or rat IGFBP-6.

Contents of the mouse/rat western-ligand blotting kit:

We routinely use biotinylated rat IGF-II as ligand! Alternatively biotinylated rat-IGF-I or biotinylated mouse IGF-I or mouse IGF-II may be used. However, mouse and rat IGF-I presumably bind weaker to mouse or rat IGFBP-6. The western-ligand blotting kit with rat IGF-II recognizes a pattern of protein bands in rat and mouse serum, that are identical with the pattern obtained with the human western-ligand blotting kit. However the bands are stained substantially stronger on a nitrocellulose membrane. The molecular weights of the stained proteins are identical to those of rat IGFBP´s described in the literature. The reagents of the kit are sufficient to stain at least 500 square-cm of blotting membrane. Each reagent of the kit is also available separately.

Our kits are supplied without substrates, you either can use your usual substrate or order either our chemiluminescent or colorimetric substrates.

We have tested other substrates successfully, as e.g. KPL, Pierce, GE healthcare, Seramun, Kem-en-tec.

A recipe for a home-made chemiluminescent substrate is available (in German Language) at:

http://www.laborjournal.de/rubric/tricks/tricks/trick81.html

Storage of reagents:

• Store all reagents protect from direct sun light.
• Reagent A: Biotinylated Ligand: The ligand can be storedat least  for one year  at 4 ° C without loss of activity.  Reconstitute with 0.5 ml of distilled water. The reconstituted ligand may be stored at least for one week at 4 °C or frozen as aliquots at - 20 °C for one year. Working dilutions should be used the same day.
• Reagent B, C, D: Concentrated Buffers: The concentrated buffers are stable for at least 6 months at 4 °C or two months at room temperature. Diluted solutions are stable for at least one week at either 4 °C or room temperature.
• Reagent E: The Streptavidin Peroxidase-Conjugate is stable for at least 6 months at 4 ° C.

Western blotting and staining of the membrane:

1. Electrophoresis

SDS-PAGE has to be done under non-reducing conditions! The samples should not be boiled, please heat to 50 °C for 3 minutes. For our experiments we have used gels of 13 to 15 % T or gradient gels (8-18 % T).

Samples: The non-radioactive western-ligand blotting methods detects IGFBP-3 in human serum in sample volumes of > 1 µl. However, for the binding proteins, that are present in low concentrations, the sample volumes should be as large as possible. Samples may  be concentrated by precipitation with ammonium sulfate (55 %, Hossenlopp et al., Anal. Biochem. 154, 138-143, 1986), lyophilisation (Grissom et al. Anal. Biochem. 212, 412-420, 1993), ultrafiltration or immuno precipitation (Fazleabas and Donnely, Anal. Biochem. 202, 40-45, 1992).
Tissue samples may be homogenised in 5 % SDS (McGuire et al. Cancer Letters 77, 25 - 32, 1994) or other detergents.
Membrane bound receptors may be solubilised with 1 - 2 % Triton X-100 (Kovacina and Roth, J. Biol. Chem. 270, 1881-1887, 1995. MacDonald and Czech, J. Biol. Chem. 260, 11357-11365, 1985). Soluble IGF-II receptors have been precipitated with 70 % ammonium sulfate (Causin et al., Biochem. J. 252, 795-799, 1988).

2. Blotting

In our experiments we used a simple capillary blotting method. The SDS-PAGE Gel is overlayed with a nitrocellulose membrane (0.22 µm) soaked in TBS ( 20 mM Tris, 145mM NaCl, pH7.4), 4 filter papers soaked with TBS and twelve dry filter papers and a stack of paper towels (2 -4 cm). A glassplate and a weight of around 2 kg are placed on top of the stack. Blotting was performed overnight.
Blotting methods with higher tranfer efficiency may improve sensitivity of the western ligand blot.
DO NOT TOUCH THE MEMBRANE WITHOUT WEARING CLEAN GLOVES!

3. Staining

For our experiments we have used at least 0.5 ml of each reagent or buffer per square-cm  of membrane surface and 0.1 ml of the substrate. If smaller quantities are used, ensure, that the membrane is covered completely!
Equipment and staining trays have to be absolutely clean and free of contaminations of heavy metals or oxidizing agents!

3.1. Preparation ofthe working solutions of reagents

All buffers and solutions should be brought to room temperature before use!
Dilute the required quantities of buffers with distilled water.
The ligand is diluted 1 : 500 in Blocking/Dilution buffer. The diluted solution should be used within 24 hours.
Dilute the Streptavidin-Peroxidase-Conjugate 1:2500 (2h incubation) to 1:5000 (overnight incubation) in Blocking/Dilution Buffer. The working solution should be used the same day.

3.2. Quenching

The membrane is placed gel side up in a glass tray with Quenching Buffer and incubated on a horizontal shaker (50 - 100 rpm) for 20 min at room temperature.

3.3. Blocking

The membrane is placed gel side up in a glass tray with Blocking Buffer and incubated on a horizontal shaker (50 - 100 rpm) for 1 hour at room temperature.

3.4. Ligand-Binding

The membrane is placed gel side up in a glass tray with ready-for-use Ligand solution and incubated on a horizontal shaker (50 - 100 rpm) for 2 hours at room temperature. The incubation may also be performed overnight, this increases sensitivity. Prevent the membrane from drying!
After the incubation, wash 5 x for 5 min with washing buffer.

3.5. Binding of the Conjugate

The membrane is placed gel side up in a glass tray with Conjugate solution and incubated on a horizontal shaker (50 - 100 rpm) for 1 hour at room temperature.
After the incubation, wash 5 x for 5 min with washing buffer.

3.6. Detection

3.6.1 Chemiluminescent detection:

In the meantime, prepare chemiluminescent substrate by mixing  equal volumes of substrate 1 and 2. The solution is stable for at least one hour at room temperature, if protected from direct sunlight.
Incubate blot by overlayering the substrate solution on the membrane for one minute. Remove blot from substrate, touch the edge to a piece of filter paper to remove excess substrate solution.
Place membrane between two sheets of clear plastic and expose to X-ray film and develop film according to the instructions of the manufacturer. Depending on the concentrations of IGFBP's exposure times from 10 sec. to 10 min. are required.
Alternatively the blot may be exposed to an instant Polaroidfilm, using a mini-camera (Amersham-Pharmacia) and a standard Polaroidfilm type 667. Using this type of film, 50 ng of the IGFBP's could be detected with an exposure time of less then ten seconds.

3.6.2.Stripping ligand blots and reprobing with antibodies

Stripping of an immunoblot with Re-Blot Plus-strong stripping solution of GE Healthcare, Piscataway, NJ and reprobing with antibodies is described in (4)

3.6.3 Colorimetric detection:

Place the membrane in a clean tray with sufficient substrate to cover the membrane and shake gently. Watch the staining process, because with high quantities of IGFBP´s, staining may occur within few minutes. Avoid direct sunlight.
After development the membrane should immediately be washed two times for five minutes with distilled water. The mebrane is the dried between sheets of filter paper and can be stored for several months, if stored dry and in the dark.
Colorimetric detection may also be done after chemiluminescent detection! Simply wash membrane after chemiluminescent detection for two minutes and the stain with the colorimetric substrate.

4.  Non-specific Staining

Non-specific staining is obtained in samples containing biotinylated molecules. If biotinylated proteins are expected, use a strip of the blot as a control, that is NOT incubated with the ligand.

5. Use of PVDF Membranes and Ultra-sensitive Chemiluminescent Substrates

We have tested the ligand blotting on PVDF membranes and/or some ultra-sensitive substrates. In some expermients we observed some background.

To avoid this background, we recommend to use 5 % non -fat dry-milk in Tris-buffered Saline (TBS) plus 0.05 % Tween-20 for blocking, washing and all incubation steps. The quenching step can be ommitted when non-fat dry-milk buffers are used. Before incubation with the chemiluminescent substrate the blot should be washed in TBS for 2 minutes.

6. Quantitative analysis

The quantitative western blot analysis is described in a aper of Laeger et al. (7). The quantitaive analysis is done as a service by Ligandis GbR.

References:

Reference:

1. Pircher et al.: A new method for the purification of bioactive insulin-like growth factor-binding protein-3. Protein Expression and Purification 71 (2010) 160–167.

Link to Abstract:

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WPJ-4YC80SN-2&_user=10&_coverDate=06%2F30%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1428943115&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f7f1e043cbca904e86719b7143f20bf9

2. Rehfeldt et al.: Increased fat mass, decreased myofiber size, and a shift to glycolytic muscle metabolism in adolescent male transgenic mice overexpressing IGFBP-2. Am J Physiol Endocrinol Metab 299: E287–E298, 2010.

Link to Abstract:

http://ajpendo.physiology.org/cgi/content/abstract/299/2/E287

3. Taferner et. al.:Purification and characterization of native human insulin-like growth factor binding protein-6. J. Cell Commun. Signal.2011.DOI 10.1007/s12079-011-0126-2.

Link to article:

http://www.springerlink.com/content/c42m052x144212n8/

4. Swain et al.: High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2. Arch Biochem Biophys. 2010 Sep 15;501(2):195-200. Epub 2010 Jun 10.

Link to Abstract:

http://www.ncbi.nlm.nih.gov/pubmed/20541521

5. Bolos et al.: ORAL ADMINISTRATION OF A GSK3 INHIBITOR INCREASES BRAIN INSULIN-LIKE GROWTH FACTOR-I LEVELS. JBC Papers in Press. Published on March 29, 2010.
http://www.jbc.org/content/early/2010/03/29/jbc.M109.096594.full.pdf+html

6. Suresh Koduru, Sivaramakrishna Yadavalli, Siva Kumar Nadimpalli: Mannose 6-phosphate Receptor (MPR 300) Proteins from Goat and Chicken Bind Human IGF-II. Published online:Biosci Rep (2006) 26:101–112.

Link to abstract:

http://www.springerlink.com/content/dj35048k148434x8/

7. Laeger et al.: Effects of Partorition and feed restriction on concentartions and sistribution of the insulin-like growth factor-binding proteins in plasma and cerebospinal fluid of dairy cows. J. Dairy Sci. 97:2875-2885. 2014.