Invitrogen

ATF4 Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell treatment to ensure that the antibody binds to the antigen stated.

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Datasheet

Protocols

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ATF4 Antibody in Immunocytochemistry (ICC/IF) — Immunocytochemistry-Immunofluorescence analysis of ATF4 in HeLa cells mock (left) and HeLa cells treated with 2 µg/mL Tunicamycin for 8hrs (right). Cells were fixed in 4% paraformaldehyde at RT for 15 min and stained using ATF4 Polyclonal Antibody (Product # PA5-27576) (Green) at a dilution of 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

ATF4 Antibody in Western Blot (WB) — CRISPR-Cas9 mediated genome editing ofATF4 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR988840_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of ATF4 was performed by loading 30 µg of HeLa Wild Type (Lane 1), treated HeLa Wild type (Lane 2), HeLa Cas9 (Lane 3), treated HeLa Cas9 (Lane 4), HeLa Cas9 cells transduced with ATF4 Lentiviral sgRNA (Lane 5) and treated HeLa Cas9 cells transduced with ATF4 and Lentiviral sgRNA (Lane 6) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with ATF4 Polyclonal Antibody (Product # PA5-27576) using 1:1,000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:5,000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toATF4. Uncharacterized band was observed in all the samples at ~38 kDa. Treatment used was 2 µg/mL tunicamycin for 8 hrs.

ATF4 Antibody in ChIP Assay (ChIP) — Enrichment of endogenous ATF4 protein at specific gene loci using Anti-ATF4 Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-ATF4 Rabbit Polyclonal Antibody (Product # PA5-27576, 3 µg) on sheared chromatin from 2 million MDA-MB-231 cells treated with Cobalt chloride (150 µM for 24 hours) using the MAGnify ChIP system kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR with PCR primer pairs over promoters of ULBP1 and ASNS (active) and SAT2 satellite repeats, SAT Alpha (inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

ATF4 Antibody — Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. A reduced signal was observed for target protein in HeLa Cas9 cell line transduced with ATF4 Lentiviral sgRNA compared to control cell line using an ATF4 Polyclonal Antibody (Product # PA5-27576). {KO}

Product Details

PA5-27576

Applications	Tested Dilution	Publications
Western Blot (WB)	1:500-1:3,000	-
Immunocytochemistry (ICC/IF)	1:100-1:1,000	-
ChIP assay (ChIP)	Assay-dependent	-

Product Specifications
Species Reactivity	Human, Mouse, Rat
Host/Isotype	Rabbit / IgG
Class	Polyclonal
Type	Antibody
Immunogen	Recombinant protein fragment corresponding to a region within amino acids 146 and 333 of Human ATF4 View immunogen
Conjugate	Unconjugated
Form	Liquid
Concentration	1.29 mg/mL
Purification	Antigen affinity chromatography
Storage buffer	PBS, pH 7, with 20% glycerol
Contains	0.025% ProClin 300
Storage conditions	Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
RRID	AB_2545052

Product Specific Information

Recommended positive controls: HeLa (4 µg/mL tunicamycin treatment for 16 hr).

Predicted reactivity: Mouse (83%), Rat (85%), Pig (89%), Sheep (86%), Rhesus Monkey (97%), Chimpanzee (99%), Bovine (90%).

Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.

Target Information

This gene encodes a transcription factor that was originally identified as a widely expressed mammalian DNA binding protein that could bind a tax-responsive enhancer element in the LTR of HTLV-1. The encoded protein was also isolated and characterized as the cAMP-response element binding protein 2 (CREB-2). The protein encoded by this gene belongs to a family of DNA-binding proteins that includes the AP-1 family of transcription factors, cAMP-response element binding proteins (CREBs) and CREB-like proteins. These transcription factors share a leucine zipper region that is involved in protein-protein interactions, located C-terminal to a stretch of basic amino acids that functions as a DNA binding domain. Two alternative transcripts encoding the same protein have been described. Two pseudogenes are located on the X chromsome at q28 in a region containing a large inverted duplication.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: Activating transcription factor 4; activating transcription factor 4 (tax-responsive enhancer element B67); activating transcription factor ATF-4; ATF 4; ATF4 protein; atf4 {ECO:0000312|RGD:621863}; C/ATF; C/EBP-related ATF; cAMP response element-binding protein 2; cAMP-dependent transcription factor ATF-4; cAMP-responsive element-binding protein 2; CREB 2; CREB-2; Cyclic AMP-dependent transcription factor ATF-4; Cyclic AMP-responsive element-binding protein 2; DNA-binding protein TAXREB67; OTTHUMP00000199130; rATF-4; tax-re; tax-responsive enhancer element B67; Tax-responsive enhancer element-binding protein 67; tax-responsive enhancer element-binding protein 67 homolog; TaxREB67; taxREB67 homolog

Gene Aliases: Atf-4; ATF4; C/ATF; CREB-2; CREB2; TAXREB67; TXREB

UniProt ID: (Human) P18848, (Rat) Q9ES19, (Mouse) Q06507

Entrez Gene ID: (Human) 468, (Rat) 79255, (Mouse) 11911

Molecular Function: DNA-binding transcription factor