Western blot analysis of extracts from COS cells, untransfected or transfected with a construct overexpressing HA-Akt, using HA-Tag (C29F4) Rabbit mAb (Biotinylated). Detection was performed with Streptavidin-HRP followed by LumiGlo® and Peroxidase #7003.
This Cell Signaling Technology (CST) antibody is conjugated to biotin under optimal conditions and tested using COS cells transfected with HA-tagged protein.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml 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.
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
HA-Tag (C29F4) Rabbit mAb (Biotinylated) detects exogenously expressed proteins containing the HA epitope tag.Species Reactivity:
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA).
Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.
The HA tag is derived from an epitope of the influenza hemagglutinin protein which has been used extensively as a general epitope tag in expression vectors (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.