Western blot analysis of ADA in, Lane 1: rat stomach tissue lysates, Lane 2: rat kidney tissue lysates, Lane 3: mouse small intestine tissue lysates. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. The membrane was blocked with 5% Non-fat Milk/ TBS for 1. 5 hour at RT. The membrane was incubated with Adenosine Deaminase Polyclonal Antibody (Product # PA5-78721) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0. 1% Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit. A specific band was detected for ADA at approximately 45KD. The expected band size for ADA is at 40KD.

Immunohistochemistry analysis of ADA on paraffin-embedded rat small intestine tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with ADA polyclonal antibody (Product# PA5-78721) with a dilution of 1 µg/mL (overnight at 4°C), and followed by biotinylated goat anti-rabbit IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.