Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 16)

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dedicated-use containers to avoid contaminating other
experiments.
Because of the high sensitivity of the chemiluminescent assay,
care should be taken to avoid alkaline phosphatase contami-
nation of the buffers and solutions used in the procedure. For
best results, the ddH
2
O used to prepare solutions should be
autoclaved or filtered prior to use in this assay.
Tris-Buffered Saline (1x TBS)
(20 mM Tris, 500 mM NaCl, pH 7.5)
If using Bio-Rad liquid concentrate 10x TBS, add 110 ml of
10x TBS to 990 ml of ddH
2
O. Label this bottle “1x TBS”.
If using solid TBS powder, follow instructions to make 1,100
ml of a 1x TBS solution. Typically, a 1x solution is 32.3 g of
TBS brought up to 1 L of solution with ddH
2
O.
Wash Solution (TTBS)
(20 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 7.5)
Add 0.8 ml of Tween 20 to 800 ml of 1x TBS. Label this bottle
“TTBS”.
Blocking Solution
(0.2% nonfat dry milk in TBS)
Add 0.2 g of nonfat dry milk to 100 ml of 1x TBS. Stir until
dissolved. Label this solution “Blocking Solution”.
Suggestions for other blockers:
casein – 0.2% in TBS, gelatin – 3% in TBS or BSA or 3% in TBS