Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with a construct expressing a 6xHis-Tag fusion protein (+), using His-Tag (D3I1O) XP® Rabbit mAb (Biotinylated).
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated His-Tag (D3I1O) XP® Rabbit mAb #12698.
Supplied in 136 mM NaCl, 2.6 mM KCl, 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 nonfat dry milk, 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: 265
His-Tag (D3I1O) XP® Rabbit mAb (Biotinylated) recognizes recombinant proteins containing the 6xHis epitope tag. The antibody recognizes the 6xHis-tag fused to either the amino or carboxy terminus of targeted proteins in transfected cells.Species Reactivity:
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues of the 6xHis epitope tag.
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.
A variety of plasmids contain DNA that encodes an amino-terminal tag consisting of six histidine (6xHis) residues followed by an extended multiple cloning site. The 6xHis tag on the expressed recombinant proteins allows for efficient coupling to Ni2+ affinity resins and purification by single step chromatography (1).
As is the case with other protein tag systems (2), this polyhistidine tag can often be cleaved at sites recognized by proteases such as thrombin and enterokinases to isolate the protein of interest (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.