Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 20)

45° angle, into the blocking solution. Gently agitate the
solution using an orbital shaker platform and incubate for
30 min to 1 hr at RT, or block overnight at 4°C.
4. Wash – decant the blocking solution and add TTBS to the
membrane. Wash for 5–10 min with gentle agitation at RT.
5. Primary antibody incubation – decant the TTBS and
add the primary antibody solution to the membrane.
Incubate 1–2 hr with gentle agitation at RT. Overnight
incubation may be preferred, since longer incubation
periods may increase the sensitivity of detection. The
optimum conditions of dilution and incubation time must
be determined experimentally. For overnight incubations,
4°C is recommended.
6. Wash – decant the primary antibody solution and add
TTBS to the membrane. Wash for 5–10 min with gentle
agitation at RT. Decant the wash solution and repeat the
wash step, with additional TTBS.
7. Second antibody conjugate incubation – decant the
TTBS and add the second antibody solution. Incubate for
30 min to 2 hr using gentle agitation at RT.
8. Wash – decant the conjugate solution and add TTBS to
the membrane. Wash for 5–10 min with gentle agitation at
RT. Decant the wash solution and repeat the wash step
two more times with additional TTBS.
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