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.
Designed for use with rabbit polyclonal and monoclonal antibodies, this affinity purified goat anti-rabbit 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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.