Alkaline Phosphatase Labeled Lectin Procedure
Western and Dot Blot Staining
1. Wash and block the tissue section or blot. EY Laboratories, Inc. recommends that 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.
2. Dilute the Alkaline Phosphatase Labeled Lectin to a concentration of 1-20 mg/ml using the recommended Buffer. 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.
3. Develop the enzyme with Dye/Substrate according to the manufacturer directions.
Notes: Inhibition of lectin binding may be accomplished by using one of the following procedures:
A. Before proceeding to Step #2 incubate the section or blot with inhibitory carbohydrate for 30-60 minutes at room temperature. Complete inhibition may not occur.
B. Preincubate the diluted Alkaline Phosphatase Labeled Lectin with the inhibitory
carbohydrate for 30-60 minutes at room temperature before applying to the section or