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.

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 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).