Anti-ATF6α antibody, mouse monoclonal (1-7)
Product code

73-500

Size

50 µg

Storage-20℃

Concentration

1.0 mg/ml

Buffer

PBS- with 50% glycerol

PurityPurified IgG fraction with protein A from hybridoma cell culture medium

Immunogen

Recombinant ATF6 (His-tagged amino-terminal fragment of ATF6)Isotype

Mouse IgG2aκ

Reactivity

Specific to human ATF6 No cross reactivity with mouse ATF6 (for mouse,BioAcademia 73-005 clone 37-1 is recommended)
This antibody is one of the two antibodies currently on market that can detectboth precursor and activated forms of ATF6 at endogenous level.

Special notes

N/AApplication

1. Western blotting (1/500-1/1,000)
2. Immunoprecipitation (1/100-1/500)
3. Immunofluorescence staining (1/100-1/500)

Background

ATF6 (activating transcription factor 6) is an endoplasmic reticulum (ER)membrane-bound transcription factor activated in response to ER stress.
When unfolded proteins accumulate in the ER, ATF6 is cleaved by regulated
intramembraneproteolysis.

Theresulting

amino-terminal

fragment

translocates to the nucleus and activates transcription by binding to ER stressresponse elements present in the promoter regions of ER stress-inducible genes
including those encoding ER chaperones and components of ER-associateddegradation. The mammalian ATF6 family consists of two closely related
homologs, ATF6 and ATF6. ATF6 but not ATF6 plays a pivotal role intranscriptional control.
The monoclonal antibody was characterized in the laboratory of Professor
Kazutoshi Mori of Kyoto University. The antibody was produced fromhybridoma cultured in serum-free medium and purified under mild conditions
by propriety chromatography processes.

Data LinkUniProtKB P18850 (ATF6A_HUMAN)

Please note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES. NOT FOR MILITARY USE.
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Data Images: 73-500 Anti-ATF6α antibody, mouse monoclonal (1-7)

Fig.1 Western blot analysis of human cell extracts using this antibody: Conversion of pATF6α(P) topATF6α(N) in DTT- or tunicamycin-treated cells.
Tm: 2 µg/ml tunicamycin (inhibitor of N-glycosylation). DTT: 1mM dithiothreitol (reducing reagent).
The asterisk denotes an unglycosylated form of pATF6α(P).
ATF6αis constitutively expressed as pATF6 (P) (~90-kDa protein), and converted to pATF6α(N)(>50-kDa protein) in ER-stressed cells.

Fig.2 IP-Western blot analysis of human cell extracts using this antibody.
ATF6αwas detected by Western blot (Input; lanes 1, 2, 7, and 8) using this antibody (No.1-7). After
immunoprecipitation (IP) with non-related IgG (IP; lanes 3, 4, 9, and 10) or this antibody (No. 1-7)
(IP; lanes 5, 6, 11, and 12), samples were subjected to SDS-PAGE and analyzed by Western blot using
this antibody (# 1-7) and anti-mouse IgG antibody (light chain specific).
Detection of pATF6α(P) and pATF6α(N) is better in IP-Western blotting than Western blotting.
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Fig.3 Immunofluorescence staining of ATF6 in HeLa cells by using anti-ATF6 antibody.
The cells were fixed with paraformaldehyde. The antibody was used at 1/100 dilution.
As the second antibody, Alexa Fluor 488 conjugated goat anti-mouse IgG antibody was used at 1/1,000dilution.
In unstressed cell ATF6α is localized in endoplasmic reticulum and upon receiving stress signal it isproteolytically processed and translocated into nucleus where it functions as transcriptional activator.

References: This antibody was described in Ref. 1 and has been used in the following publications.
1. Mori K .Divest yourself of a preconceived idea: transcription factor ATF6 is not a soluble protein!
Mol Biol Cell 21: 11435-8 (2010) PMID: 20219975 Review article
2. Maiuolo J et al. Selective activation of the transcription factor ATF6 mediates endoplasmic
reticulum proliferation triggered by a membrane protein. PNAS 108: 7832-7 (2011) PMID:21521793 WB (human)
3. Amyot J et al. Binding of activating transcription factor 6 to the A5/Core of the rat insulin II genepromoter does not mediate its transcriptional repression. J Mol Endocrinol. 2011 Sep30;47(3):27383. PMID:21821716 WB (human)
4. Higa A. et al. Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior
gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. J Biol Chem. 2011 Dec
30;286(52):44855-68 PMID:22025610 WB (human)
5. Bouchecareilh M et al. Small GTPase Signaling and the Unfolded Protein Response. Methods inEnzymology Vol. 491; p. 343-360 (2011). PMID:21329809 WB (human)
6. Kondo S. et al. Activation of OASIS family, ER stress transducers, is dependent on its stabilization
Cell Death and Differentiation (2012) 19, 1939–1949. PMID: 22705851 WB (human)
7. Chiang WC et al. Selective Activation of ATF6 and PERK Endoplasmic Reticulum Stress Signaling
Pathways Prevent Mutant Rhodopsin Accumulation. Invest Ophthalmol Vis Sci. 2012 Oct; 53(11):7159–7166. PMID:22956602 WB (human)
8. Yi P et al. Sorafenib-mediated targeting of the AAA⁺ ATPase p97/VCP leads to disruption of
the secretorypathway, endoplasmic reticulum stress,and hepatocellular cancer cell death.

Mol Cancer Ther. 2012 Dec;11(12):2610-20. PMID: 23041544 WB (human)
9. Shoulders MD et al. Stress-independent activation of XBP1s and/or ATF6 reveals three functionallydiverse ERproteostasis environments. Cell Rep. 2013 Apr 25;3(4):1279-92. PMID:23583182 WB
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10.Kitai Y et al. Membrane lipid saturation activates IRE1α without inducing clustering. Genes
Cells. 2013 Sep;18(9):798-809. PMID:23803178 WB (human)
11. Higa A. et al. Endoplasmic reticulum stress-activated transcription factor ATF6α requires thedisulfide isomerase PDIA5 to modulate chemoresistance. Mol Cell Biol. 2014 May;34(10):1839-49.
PMID: 24636989 WB (human)
12. Miyata S. et al. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing
Sterol Regulatory Element-binding Protein (SREBP) Activation. J Biol Chem. 2015 Aug
14;290(33):20565-79. PMID: 26140926 WB (human)
13. Plate L et al. Small molecule proteostasis regulators that reprogram the ER to reduce
extracellular protein aggregation. Elife. 2016 Jul 20;5. pii: e15550. PMID:27435961 WB (human)
Protocol for ATF6α analysis using anti-human ATF6α monoclonal antibody
Both endogenous precursor ATF6 , pATF6 (P), and its cleaved product, pATF6α(N), can be detected
in human cells such as HEK293T, HEK293 and HeLa cells by western blot analysis using anti-human
ATF6α monoclonal antibody clone 1-7 (Fig. 1), according to the procedures described below. Clarityof the results may depend on cell types and culture conditions. If clear results could not be obtainedby western blot analysis as seen with HeLa and HEK293 cells (Fig.1 right panel), it is worth while
trying immunoprecipitation followed by western blot analysis according to the procedures describedbelow.
Western blotting
SDS-sample buffer: 50 mM Tris/HCl, pH6.8, containing 2% SDS, (100 mM DTT), 10% glycerol andBPBPBST: PBS containing 0.1% Tween 20 Blocking buffer: PBS containing 0.1% Tween 20 and 5% skim
milk
Sample Preparation (for HeLa or HEK293 cells cultured in 6cm dish)(1) Wash cells with ice-cold PBS.
(2) Scrape cells in 500 l of ice-cold PBS (+ protease inhibitor cocktail and 10 M MG132) 2 times andcollect cells by centrifugation at 5,000 rpm for 2 min.
(3) Lyse cells directly in 100 l of SDS-sample buffer without reducing reagent (+ protease inhibitor
cocktail and 10 M MG132).
(4) Voltex mix vigorously.
(5) Boil the lysate for 5 min and voltex well.
(6) If the lysate is still viscous, boil again and voltex mix vigorously.
(7) Centrifuge at 14,000 rpm for 2 min.
(8) Determine protein concentration using BCA protein assay kit.
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SDS-PAGE and incubation with antibody(9) Add one-tenth volume of 1 M DTT and boil for 5 min.
(10) Subject 50 g of the lysate to 8% SDS-PAGE.
(11) Transfer to nitrocellulose membrane (such as Hybond-ECL, GE Healthcare).
(12) Incubate the membrane in Blocking buffer overnight at 4˚C.
(13) Incubate the membrane with primary antibody diluted in Blocking buffer (1:500-1:1000) for 1 hat room temperature or overnight at 4˚C. Wash the membrane 3 times each for 5 min with PBST.
(14) Incubate the membrane with HRP-conjugated secondary antibody for 1 h at room temperature.
We recommend “ECL anti-mouse IgG, Horseradish Peroxidase linked F(ab’)2 fragment” (GEHealthcare NA9310V-1ML) or “Peroxidase-conjugated AffniPure Goat Anti-Mouse IgG, Light
Chain Specific” (Jackson ImmunoReseach 115-035-174).
(15) Wash the membrane 3 times each for 5 min with PBST.
Detect signals using an appropriate luminescent reagent.
Immunoprecipitation(For HeLa or HEK293 cells cultured in 6 cm dish)Lysis buffer: 50 mM Tris-HCl pH7.5, containing 150 mM NaCl, 1% NP-40, protease inhibitor cocktailand 10 M MG132)(1)

Wash cells with ice-cold PBS, suspend them in 400 l of Lysis buffer and stand on ice for 10 min.

(2)

Clear the lysate by centrifugation at 14,000rpm for 10 min at 4˚C and transfer 300 l of thesupernatant to a new tube.

(3)

Add anti-ATF6 antibody (1-3 g) into the supernatant and incubate with gentle rotation for 2
h〜overnight at 4˚C.

(4)Add 30 l of the 50% slurry of ProteinG-Sepharose suspended in Lysis buffer into the tube andincubate with gentle rotation for 1 h at 4˚C.

(5)Wash the Sepharose beads 2 times with Lysis buffer.

(6)

Wash the Sepharose beads with PBS.

(7)

Resuspend the Sepharose beads in 30 l of SDS-sample buffer containing 100 mM DTT. Use 10
l aliquot of the sample for Western blot analysis.

*Critical point■
In IP-western blot analysis, use a light chain specific anti-mouse IgG antibody as a secondaryantibody. “Peroxidase-conjugated AffniPure Goat Anti-Mouse IgG, Light Chain Specific”(Jackson ImmunoReseach 115-035-174) is recommended.

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