Revision 7

#3724Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, 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 10 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
Immunohistochemistry (Paraffin) 1:800 - 1:3200
Immunofluorescence (Immunocytochemistry) 1:800 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:800 - 1:1600
Chromatin IP 1:50

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.

For a carrier free (BSA and azide free) version of this product see product #86124.

Specificity / Sensitivity

HA-Tag (C29F4) Rabbit mAb detects exogenously expressed proteins containing the HA epitope tag. The antibody may cross-react with a protein of unknown origin ~100kDa.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA).

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.

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).

  1. Field, J. et al. (1988) Mol Cell Biol 8, 2159-65.

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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) 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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 7
#3724

HA-Tag (C29F4) Rabbit mAb

Western Blotting Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Western blot analysis of extracts from HeLa cells, untransfected or transfected with either HA-FoxO4 or HA-Akt3, using HA-Tag (C29F4) Rabbit mAb.
Western Blotting Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from COS-7 cells, transfected with a construct expressing HA-tagged Stat3 using HA-Tag (C29F4) Rabbit mAb #3724. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Immunoprecipitation of HA-FoxO4 tag protein from 293T transfected cell extracts. Lane 1 is 10% input, lane 2 is HA-Tag (C29F4) Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot was performed using HA-Tag (C29F4) Rabbit mAb. A conformation specific secondary antibody was used to avoid reactivity with IgG.
Immunohistochemistry Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded COS cells, HA-Tag transfected (left) or untransfected (right), using HA-Tag (C29F4) Rabbit mAb.
Immunofluorescence Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of COS cells, transfected with an HA-tagged protein (left) or mock-transfected (right), using HA-Tag (C29F4) Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® (fluorescent DNA dye).
Flow Cytometry Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
Flow cytometric analysis of 293T cells untransfected (blue) or transfected with HA-tagged Akt (green), using HA-Tag (C29F4) Rabbit mAb (solid lines) or concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines).

Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Chromatin Immunoprecipitation Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
293T cells were either untransfected (left panel) or transfected with an HA-tagged human CBP construct (right panel), then treated with Forskolin #3828 (30 µM) . Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and HA-Tag (C29F4) Rabbit mAb, CBP (D9B6) Rabbit mAb, or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.