Western-Ligand Blotting Kit for Mouse/Rat IGFBP´s

Western-Ligand Blotting Kit for Mouse/Rat IGFBP´s

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 IGFBPs 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.

Reagent Description Quantity Product Code Price Euro
A 0.5 ml Biotinylated ligand: biotinylated rat IGF-II, dilute 1 : 100 to 1 : 500 10 µg BioRATIGF2-10 420.- 
B Quenching Buffer, 2-fold conc. 125 ml  QB-125 15.50 
C Blocking/Dilution Buffer, 10 - fold conc. 75 ml BDB-75 50.- 
D Washing Buffer, 20-fold conc.  125 ml Product Code: WB-125 35.- 
E Streptavidin-POD-Conjugate,
dilute 1:2500 - 1:5000		 125 µl Product Code: SPCON 67.- 
  Detailed working instructions   Application Note IGF005  
  Rat/Mouse Western-ligand Blot 1 Kit Product Code: rat/mouse-WLB 535.-

Our kits are supplied without substrates, you can use your usual substrate.

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

The colorimetric substrate can be used after the chemiluminescent substrate to give a permanent record. The membrane is simply washed for 1 min and the incubated with the colorimetric substrate. The sensitivity of the colorimetric substrate is similar to the chemiluminescent substrate, but incubation times are longer!

NOTES ON USE OF YOUR OWN BUFFERS

We have developped a buffer system, giving an optimal sensitivity on nitrocellulose membranes in our hands and having a prolonged stability, even at room temperature. Other buffers may propably be used, with a few limitations:

  1. Take care of the quality of bovine serum albumin (BSA), if you use it for blocking and/or dilution. Many BSA preparations contain traces of IGFBPs, that may affect the quality (Background/signal ratio) of the WLBs.
  2. In literature it is described that milk contains biotinylated proteins, that might affect the quality (background/signal ratio) of the WLB's. We have tested non-fat dry milk from different suppliers and from our local supermarket and found no biotinylated proteins.

Reference:

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

 

Rev. 2013-02

     Datasheet for Western-Ligand Blotting Kit for Mouse/Rat IGFBP´s

    Category: Blotting Kits
    Itemnumber: Product Code: rat/mouse-WLB
    Price: EUR 462.00
    Quantity:

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