Table 2.12.2.

Making Dilutions of the Primary Antibody for a Titration^a

Final concentration	Final volume	Antibody (μl)	Buffer added (KPBS + 0.4% Triton X-100)
1:1000	3 ml	3 μl of 1:1	2997 μl
1:3000	2 ml	666.7 μl of 1:1000	1333.3 μl
1:10,000	2 ml	200 μl of 1:1000	1800 μl
1:30,000	2 ml	66.7 μl of 1:1000	1933.3 μl
1:100,000	2 ml	20 μl of 1:1000	1980 μl
1:300,000	2 ml	6.7 μl of 1:1000	1993.3 μl

^aThe chart assumes that the starting material is pure serum (1:1). Immunoglobulins are normally present in blood at a concentration of ~100 mg/ml. If purchased antibodies are provided as mg or μg rather than a volume, prepare a starting concentration of 100 mg/ml in distilled water and term that solution 1:1. Storage of stock solutions is at −70°C (small aliquots can be prepared and stored this way, if desired). When using the antibodies on a regular basis, dilute an aliquot of the stock solution to either 1:10 or 1:100 in a glycerol solution consisting of 1 g bovine serum albumin, 50 g 100% glycerol, and 0.05 g sodium azide in 100 ml of 0.05 M KPBS, and keep this solution at −20°C.