Horseradish Peroxidase (HRP) Labeled Lectin Procedure

1. Endogenous peroxidase activity must be blocked prior to incubating with the lectin conjugate.

Pretreat the section or blot with 0.6% hydrogen peroxide in methanol for 20 min. at room temperature. After blocking, briefly rinse with the recommended Buffer.

2. Wash and block the tissue section or blot. EY Laboratories, Inc. recommends that a purified Bovine Serum Albumin (BSA) or defatted milk powder be used for blocking to prevent non-specific binding.  Do not use serum products, they contain glycoproteins which may lead to high levels of non specific background.

3. Dilute the HRP Labeled Lectin to a concentration of 1-20 mg/ml using the recommended Buffer (sodium azide-free). Incubate the section or blot for 30-90 minutes at room temperature in a moist chamber.  Slightly longer incubation time may be required if the incubation is done at 2-8ºC.  Rinse 3 times, 5 minutes each time with the recom­mended Buffer.

4. Develop the enzyme with Dye/Substrate according to the manufacturer’s directions.

NOTE: Inhibition of lectin binding may be accomplished by using one of the following procedures:

A. Before proceeding to Step #3 incubate the lectin treated section or blot with the
inhibitory carbohydrate for 30-60 minutes at room temperature.  Complete inhibition
may not occur.

B. Preincubate the diluted HRP Labeled Lectin with the inhibitory carbohydrate for 30-
60 minutes at room temperature before applying to the section or blot.