Western blot analysis of extracts from HeLa cells, untreated or treated with Paclitaxel #9807, using Phospho-Histone H3 (Ser10) (D2C8) Rabbit mAb (Biotinylated) #3642 and Streptavidin-HRP #3999 for detection.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 2 mg/ml BSA, and 50% glycerol. Store at –20°C.
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.
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.
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.
* 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 June 2016
Protocol Id: 266
This Cell Signaling Technology product is useful for the detection of biotinylated proteins (1,2). Conjugation of horseradish peroxidase (HRP) to streptavidin is obtained by cross linking the amino groups on streptavidin with the carbohydrate groups on HRP.
Streptavidin is a 53 kDa homotetramer isolated from Streptomyces avidinii for use in isolation and detection bioassays (3). Each streptavidin subunit forms high affinity non-covalent bonds with the vitamin biotin. Because of its strong non-covalent interaction with biotin, streptavidin can be used to detect and isolate biotinylated proteins (1,2).
Chemiluminescent detection systems have emerged as the best all-around detection method for use with western blots and ELISA. These detection assays eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives. Streptavidin-HRP is used with biotinylated proteins and specific chemiluminescent substrates to generate light signal. Streptavidin-HRP conjugates have a very high turnover rate, coupling high sensitivity with short reaction times.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.