For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Two-color western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. Overlap of epitopes may cause interference and should be considered in two color western blots. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) is recommended to verify electrotransfer and to determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
CRITICAL STEP: Do not include Tween® 20 in blocking buffer (Section A, Step 8).
Drain membrane of excess TBST and allow to dry.
CRITICAL STEP: Membrane must be dry for fluorescent staining.
posted May 2008
revised June 2016