Western blot analysis of extracts from COS cells, untransfected (-) or transfected with DYKDDDDK-tagged Fox03 (+), using DYKDDDDK Tag Antibody (HRP Conjugate).
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.
* 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: 264
DYKDDDDK Tag Antibody (HRP Conjugate) detects exogenously expressed DYKDDDDK proteins in cells. The antibody recognizes the DYKDDDDK peptide (the same epitope recognized by Sigma's Anti-FLAG® antibodies) fused to either the amino or carboxy terminus of targeted proteins. The binding specificity of this antibody is NOT dependent on the presence of divalent metal cations.Species Reactivity:
All Species Expected
Polyclonal antibodies are produced by immunizing animals with a synthetic DYKDDDDK peptide. Antibodies are purified by protein A and peptide affinity chromatography. The antibody was conjugated to HRP under optimal conditions.
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 DYKDDDDK peptide has been used extensively as a general epitope tag in expression vectors. This peptide can be expressed and detected with the protein of interest as an amino-terminal or carboxy-terminal fusion (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Anti-FLAG is a registered trademark of Sigma-Aldrich Biotechnology.