Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #2500: Human EGF antiserum (Rabbit) for RIA, IHC, WB

Anti-Human EGF is an antiserum specific for EGF. The antiserum was obtained from rabbits immunized with recombinant human EGF. Supplied in lyophilized form, each vial contains the equivalent of 5µl of serum. The antiserum is stable for at least 2 years* at 2-4°C in a lyophilized state.

* Longer term stablilty is expected to be demonstrated.

1. Recommended handling for use in Radioimmunoassay (RIA):

For radioimmunoassay we recommend the antiserum be used at a final dilution of 1/500,000. At this dilution the antiserum binds 30% of [¹²⁵I]-labelled human EGF tracer (80 Ci/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 1.4 ng/ml of human EGF and the limit of detection is approximately 0.3 ng/ml.

Procedure:

Dissolve the vial contents in 250 µl of RIA buffer (30 mM sodium phosphate pH 7.5 containing 10mM EDTA, 0.2 g/l protamine sulphate, 0.5 ml Tween-20 and 0.25 g/l sodium azide) to give an antiserum stock of 1/50. This 1/50 stock solution may be aliquoted and stored at -20°C. The 1/50 stock is further diluted in the same buffer to yield a 1/100,000 working solution. Working solutions are stable for 5 days at 4°C.

50 µl of the 1/100,000 working solution is added to each RIA tube (except the antibody-negative "blanks") in a final incubation volume of 0.25 ml giving a final dilution of antiserum of 1/500,000. Each tube also contains approximately 20,000 cpm of fresh [¹²⁵I]-labelled human EGF (80 Ci/g). Bound radioactivity is precipitated using conventional centrifugation procedure after addition of anti-rabbit IgG, rabbit IgG and chilled PEG.

See Protocols:
#3500 Procedure for the Iodination of EGF / Betacellulin Peptides.

#3501 Determination of EGF / Betacellulin by Radioimmunoassay (RIA).

Results:

The relative potencies of the following peptides at inhibiting the binding of [¹²⁵I]-labelled human EGF to this antiserum were found to be:


human EGF
100%
LONG®EGF
56%
mouse EGF
< 0.01%
LONG®R³IGF-I
< 0.01%
human TGFα
< 0.01%

2. Recommended handling for use in immunoblotting:

Novozymes GroPep's anti-human EGF antiserum has been successfully used for western immunoblotting. We recommend the antiserum be used at a final dilution of 1/1,000 to 1/5,000.

See Protocol:
#3520 Procedure for Western Immunoblotting of EGF / Betacellulin.


3. Recommended handling for use in immunohistochemistry (IHC):

Novozymes GroPep's anti-human EGF antiserum has been successfully used in immunohistochemistry in paraffin sections using immunoperoxidase staining.

See Protocol:
#3510 Immunohistochemical (IHC) detection of human EGF in paraffin sections by Immunoperoxidase staining.


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