His-Tag (D3I1O) XP® Rabbit mAb (#12698) Datasheet with Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:

WB, W-S, IP, IF-IC, FC-FP, ChIP

REACTIVITY:

All

SENSITIVITY:

Transfected Only

MW (kDa):

Source/Isotype:

Rabbit IgG

Product Information

Product Usage Information

For optimal ChIP results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x 10⁶ cells) per IP. This antibody has been validated using SimpleChIP^® Enzymatic Chromatin IP Kits.

Application	Dilution
Western Blotting	1:1000
Simple Western™	1:50 - 1:250
Immunoprecipitation	1:50
Immunofluorescence (Immunocytochemistry)	1:400
Flow Cytometry (Fixed/Permeabilized)	1:800
Chromatin IP	1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

His-Tag (D3I1O) XP^® Rabbit mAb 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

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues of the 6xHis epitope tag.

Background

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).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: 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.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Alexa Fluor is a registered trademark of Life Technologies Corporation.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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