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LAB 4 — Western Blotting OBJECTIVES 1. To complete the process of Western blotting: Washes, secondary antibody incubation, washes, detection. 2. Use a gel-doe system to detect the presence of the secondary antibody bound to the primary antibody which is bound to Alkaline Phosphatase. Introduction In the previous lab, a general detection of the protein we isolated from the IP was viewed with Ponceau S stain. This week we will attempt to specifically detect Alkaline Phosphate with a Western blot targeting the FLAG tag. The membrane that was used in the previous lab has been incubated with primary antibody targeting the FLAG epitope since yesterday evening. The membrane must now be washed, incuabted with a secondary antibody which targets the species in which the primary antibody was generated, washed again, and then detected. The detection will be performed using a modified alkaline phosphatase (AP) method. The secondary antibody has AP bound to it, which will react with a colourimetric substrate solution to produce a purple product wherever the secondary antibody is located on the membrane. This will result in the tagged protein turning a purple colour. Solutions prepared by the Demonstrator 1. Primary antibody solution, 5% skim milk in TBST with 1:1500 dilution of primary antibody (Rabbit anti-FLAG (RbaFLAG)) (Invitrogen), 2. Western Development (BCIP/NBT) reagent (6ml aliquots): 1-Step™ NBT/BCIP Substrate Solution (Thermo Fisher) Solutions to be made by the students 1. TBST (1x) (Ix TBS with 0.1% Tween 20) 2. Secondary antibody solution (5% skim milk in 1x TBST with 1:2 P (000 diluti f secondary antibody) (Goat anti-Rabbit AP) (CedarLane) eae - Detecting PhoA 42 
