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.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 7
#3724

HA-Tag (C29F4) Rabbit mAb

Western Blotting Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
使用 HA-Tag (C29F4) Rabbit mAb 对来自未转染或用 HA-FoxO4 或 HA-Akt3 转染的 HeLa 细胞提取物进行蛋白质印迹分析。
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. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:50 和 1:250 时的单一靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:50(蓝线)和 1:250(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
Immunoprecipitation Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
对 293T 转染细胞提取物中的 HA-FoxO4 标签蛋白进行免疫沉淀分析。泳道 1 为 10% 输入,泳道 2 为 HA-Tag (C29F4) Rabbit mAb,泳道 3 为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900。使用 HA-Tag (C29F4) Rabbit mAb 进行蛋白质印迹分析。构象特异性二抗用于避免与 IgG 的反应性。
Immunohistochemistry Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
使用 HA-Tag (C29F4) Rabbit mAb 对转染 HA 标签(左图)或未转染(右图)的石蜡包埋的 COS 细胞进行免疫组织化学分析。
Immunofluorescence Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
使用 HA-Tag (C29F4) Rabbit mAb(绿色),对用加 HA 标签的蛋白质转染(左)或模拟转染(右)的 COS 细胞进行共聚焦免疫荧光分析。肌动蛋白纤丝已用 DY-554 phalloidin(红色)标记。蓝色伪彩 = DRAQ5®(DNA 荧光染料)。
Flow Cytometry Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
使用 HA-Tag (C29F4) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对未转染(蓝色)或转染带 HA 标签的 Akt(绿色)的 293T 细胞进行流式细胞分析。

Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。

Chromatin Immunoprecipitation Image 1: HA-Tag (C29F4) Rabbit mAb Expand Image
293T 细胞未转染(左小图)或用加 HA 标签的人 CBP 表达载体(右小图)转染,随后用 Forskolin #3828 (30 µM) 处理。使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对细胞的交联染色质,在加入 HA-Tag (C29F4) Rabbit mAb、CBP (D9B6) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用人 ALS2 外显子 1 引物、SimpleChIP® Human NR4A3 Promoter Primers #4829 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。