Revision 11

#11815Store 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, IP, IF-IC, ChIP, ChIP-seq, C&R, C&T

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

49

Source/Isotype:

Rabbit IgG

UniProt ID:

#P18848

Entrez-Gene Id:

468

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 2.5 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.

The CUT&RUN dilution was determined using the CUT&RUN Assay Kit #86652.

The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Chromatin IP 1:200
Chromatin IP-seq 1:200
CUT&RUN 1:200
CUT&Tag 1:200

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

ATF-4 (D4B8) Rabbit mAb recognizes endogenous levels of total ATF-4 protein.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ATF-4 protein.

Background

ATF-4, an activating transcription factor/cAMP-response element-binding protein family member, functions in the PERK and eIF2α ER stress responsive pathway (1-3). ER stress represses the translation of the majority of mRNAs, but selectively stimulates the translation of certain mRNAs including that of ATF-4 (2). Induced expression of ATF-4 increases the expression of genes critical for the recovery from ER stress (4).

  1. Fawcett, T.W. et al. (1999) Biochem J 339 (Pt 1), 135-41.
  2. Harding, H.P. et al. (2000) Mol Cell 6, 1099-108.
  3. van Huizen, R. et al. (2003) J Biol Chem 278, 15558-64.
  4. Yusta, B. et al. (2006) Cell Metab 4, 391-406.

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 IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag

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.
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 11
#11815

ATF-4 (D4B8) Rabbit mAb

Western Blotting Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
Western blot analysis of extracts from 293 and HeLa cells, untreated (-) or tunicamycin-treated (2 μg/ml, 8 hr; +), using ATF-4 (D4B8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
Immunoprecipitation of ATF-4 from extracts of 293 cells, treated with tunicamycin (2 μg/ml, 8 hr), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ATF-4 (D4B8) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ATF-4 (D4B8) Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
Immunofluorescence Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or tunicamycin-treated (2 μg/ml, 8 hr; right), using ATF-4 (D4B8) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
Chromatin Immunoprecipitation Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Ddit3/CHOP, a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 2: ATF-4 (D4B8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 10 (upper), including Ddit3/CHOP (lower), a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: ATF-4 (D4B8) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight, and ATF-4 (D4B8) 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 SimpleChIP® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.
CUT and RUN Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ASNS, a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: ATF-4 (D4B8) Rabbit mAb Expand Image
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 7 (upper), including ASNS (lower), a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: ATF-4 (D4B8) Rabbit mAb Expand Image
CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and either ATF-4 (D4B8) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human TRB3 exon 1 primer, human ASNS exon 1 primer 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.
CUT and Tag Image 1: ATF-4 (D4B8) Rabbit mAb Expand Image
CUT&Tag was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across Ddit3/CHOP gene, a known target gene of ATF4 (see our ChIP-qPCR figure).
CUT and Tag Image 2: ATF-4 (D4B8) Rabbit mAb Expand Image
CUT&Tag was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 12 (upper), including Ddit3/CHOP (lower), a known target gene of ATF-4 (see our ChIP-qPCR figure).