Biotinylated Protein Ladder Detection Pack

(a) Thaw protein ladder on ice. Mix well and aliquot the desired amount of the protein ladder (10 µl for mini-gels and 20 µl for full length gels) into a separate tube.

(b) Heat the ladder to 95-100ºC for 2 minutes.

(c) After a quick microcentrifuge spin, load directly onto gel. To ensure uniform mobility, load an equal volume of 1X Reducing SDS Loading Buffer into any unused wells.

(d) Peform Chemiluminescent detection according to manufacturer's instructions except, at the stage of secondary antibody incubation with the blot, concurrently add the anti-biotin HRP-linked antibody (provided) at a dilution of 1:1000. Incubate for 1 hour at room temperature with gentle agitation.

(e) Proceed as usual with the detection procedure

Biotinylated Protein Ladder: Store at -20°C.
Anti-biotin HRP-linked Antibody: Store at -20°C.

The Anti-biotin, HRP-linked Antibody may cross react with endogenously expressed biotinylated proteins. Careful titration may be needed.

Biotinylated protein ladder detection pack is designed to detect the molecular weight ladders on Western blots when using the horseradish peroxidase (HRP) based Western detection system. The pack contains Biotinylated Protein Ladder and Anti-biotin, HRP-linked Antibody. The molecular weight ladder is a mixture of purified proteins covalently coupled to biotin that resolve to 10 bands that have a size range of 9-200 kDa. The anti-biotin antibody is used to detect biotinylated protein ladders on Western blots. The pack is optimized for chemiluminescent Western detection procedures.