Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #5001: Handling Of GroPep Bioreagents Goat anti-rabbit secondary antibody

Goat anti-rabbit secondary antibody is derived from serum harvested from goats that have been immunised against pure rabbit gamma globulin. The antibody preparation is a 0 - 40% saturated ammonium sulphate cut of the original serum (contains the IgG fraction). Each vial contains 76 mg of lyophilized protein, and is sufficient for approximately 250 RIA assay tubes. The antibody is stable for at least 2 years* at 2 - 4 °C in a lyophilized state.

Vial reconstitution:

The vial contents are under a slight vacuum. Care should be taken when opening the vial to equilibrate the contents with ambient pressure to avoid losses. An air-filled syringe may be introduced through the bung to equalize the pressure before opening.

The antibody preparation should be reconstituted in an RIA buffer (30 mM NaH₂P0₄, 0.2% protamine sulphate, 10 mM EDTA, 0.2% NaN₃, 0.05% Tween-20, pH 7.5) or an equivalent neutral pH buffer in which the preparation is to be used.

To ensure optimum recovery of the antibody preparation it is recommended that the contents of the vial be thoroughly dissolved in the vial using 1.5 ml of the buffer solution before transferring the contents to any other container for further dilution. Please note that the preparation will reconstitute more readily if left to stand for a time following the addition of buffer. Solutions of antibody can be stored for at least 3 months* at -20°C or -80°C.

Please note that multiple freeze-thawing cycles may damage the activity of the product and therefore should be avoided.

(*Longer term stability tests of lyophilized and frozen stock solutions are continuing and extended stability is expected to be demonstrated).

Recommended concentration of secondary antibody preparation for use in a double antibody radioimmunoassay:

Please note that it is essential to titre both the secondary antibody (goat anti-rabbit antiserum) and the rabbit gamma globulin in the assay system to determine the concentrations of both reagents required to achieve optimum precipitation. The following recommendations should be taken as a guide only.

1. Reconstitute the contents of the vial of goat anti-rabbit antiserum as indicated above (i.e. dissolve lyophilized antibody pellet in 1.5 ml of RIA buffer or equivalent, let stand and then mix thoroughly before transferring contents from the vial).

2. Transfer the 1.5 ml of antibody solution from the vial and place in a tube or container containing an additional 11 ml of RIA buffer or equivalent. (Therefore, the contents of each vial are dissolved in a total of 12.5 ml of assay buffer before use in an RIA).

3. Add 50 µl of this working solution of secondary antibody to each RIA tube together with a previously determined amount of rabbit gamma globulin (IgG).

4. An alternative dilution procedure can be used as long as the final concentration of secondary antibody has been titred in the user's assay system.

See Protocol:
#3002 Determination of IGF-I or IGF-II in a Range of Species by Radioimmunoassay (RIA).

* NOT FOR USE IN HUMANS


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