After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
Affinity purified horse anti-mouse IgG (heavy and light chain) antibody is conjugated to horseradish peroxidase(HRP) for chemiluminescent detection. This product is thoroughly validated with CST primary antibodies and will work optimally with the CST western immunoblotting protocol, ensuring accurate and reproducible results.
Recommended Antibody Dilutions:
20X LumiGLO® Reagent and 20X Peroxide #7003 1:1K–1:3K
SignalFireTM ECL Reagent #6883 1:1K–1:3K
SignalFireTM Plus ECL Reagent #12630 1:5K-1:15K
SignalFireTM Elite ECL Reagent #12757 1:10K-1:20K
Supplied in 10mM sodium HEPES (pH 7.5), 150 mM NaCl, 2 mg/ml bovine serum albumin (BSA) and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
NOTE: Please refer to the primary antibody datasheet or product webpage for recommended primary antibody dilution buffer and recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) or smaller membrane; for larger membranes, adjust volumes accordingly..
Proceed to one of the following specific set of steps depending on the primary antibody used.
For Unconjugated Primary Antibodies
For HRP Conjugated Primary Antibodies
For Biotinylated Primary Antibodies
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately after incubation.
Protocol Id: 2064
Chemiluminescence systems have emerged as the best all-around method for western blot detection. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives, and because results are generated on film, it is possible to record and store data permanently. Blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. HRP-conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times.
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