Atf6 - 73505ex.20100921

MONOCLONAL ANTIBODY
For research use only. Not for clinical diagnosis.
Catalog No. BAM-73-505-EX
Anti-ATF6 antibody, (37-1)

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
intramembrane proteolysis. The resulting amino-terminal fragment translocates to the nucleus and activates
transcription by binding to ER stress-response elements present in the promoter regions of ER stress-inducible
genes including those encoding ER chaperones and components of ER-associated degradation. ATF6 consists of
two closely related factors, ATF6 and ATF6, in mammals. ATF6 but not ATF6 plays a pivotal role in
transcriptional control.
The monoclonal antibody was characterized in the laboratory of Professor Kazutoshi Mori of Kyoto University. The
antibody was produced from hybridoma cultured in serum-free medium and purified under mild conditions by
propriety chromatography processes.
Product type
Host
Source
Form
Volume
Concentration
Specificity
Antigen
Clone
Isotype
Application notes
Primary antibodies
Mouse
Culture Supernatant
Liquid
Purified monoclonal antibody (IgG) 1mg/ml in PBS, 50% glycerol, filter-sterilized
100 g
ATF6
Recombinant ATF6 (amino-terminal fragment of ATF6 fused to GST)
Epitope: not determined
37-1
Mouse IgG1
WB, Immunoprecipitation (IP) (less efficient than clone1-7)

This antibody does not work for immunofluorescence analyses.
Recommended use
Recommended dilutions
Cross reactivity
Storage
References
Optimal dilutions/concentrations should be determined by the end user.

Data Link: Swiss-Prot [P18850] (human ATF6 alpha)
human and mouse ATF6
However, clone 1-7 antibody (#BAM-73-500-EX) is recommended for human cells.
-20C (long period, -70C)
1) Hai T, et al., (1989) "Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able
to selectively form DNA-binding heterodimers." Genes Dev 3: 2083-2090 [PMID: 2516827]
2) Haze K, et al., (1999) "Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and
activated by proteolysis in response to endoplasmic reticulum stress". Mol Biol Cell 10: 3787-3799 [PMID:
10564271]
3) Yamamoto K. et al., (2007) "Transcriptional induction of mammalian ER quality control proteins is mediated by
single or combined action of ATF6 and XBP1". Dev. Cell 13: 365-376 [PMID: 17765680]
www.cosmobio.co.jp
Protocol for ATF6 analysis using anti-human ATF6 monoclonal antibody (37-1)
Both endogenous precursor ATF6, pATF6(P), and its cleaved product, pATF6(N), can be detected in human
cells such as HeLa cells by western blot analysis using anti-human ATF6 monoclonal antibody clone 37-1 (Fig. 1),
according to the procedures described below.
As clone 37-1 cross reacts with mouse ATF6, both endogenous precursor ATF6, pATF6(P), and its cleaved
product, pATF6(N), can be detected in mouse cells such as NIH3T3 cells by western blot analysis (Fig. 2),
according to the procedures described below.
Fig.1 Western blot analysis of human cell extracts using this antibody: Conversion of pATF6(P)
to pATF6(N) in DTT- or tunicamycin-treated cells.
1) untreated
2) DTT: 1mM dithiothreitol (reducing reagent) for 1 h.
3) Tm: 2 g/ml tunicamycin (inhibitor of N-glycosylation) for 3 h.
4) Tm: 2 g/ml tunicamycin (inhibitor of N-glycosylation) for 7 h.
The asterisk denotes an unglycosylated form of pATF6(P).
ATF6 is constitutively expressed as pATF6(P) (~90-kDa protein), and converted to pATF6(N) (>50-kDa protein)
in ER-stressed cells.
10 min.
exposure
1 min.
exposure
with Can Get Signal
Fig.2 Western blot analysis of mouse cell extracts using this antibody: Conversion of pATF6(P)
to pATF6(N) in DTT- or tunicamycin-treated cells.
1) untreated.
2) DTT: 1mM dithiothreitol for 1 h.
3) Tm: 2 g/ml tunicamycin for 3 h.
4) Tm: 2 g/ml tunicamycin for 7 h.
The asterisk denotes an unglycosylated form of pATF6(P).
ATF6 is constitutively expressed as pATF6(P) (~90-kDa protein), and converted to pATF6(N) (>50-kDa protein)
in ER-stressed cells.
www.cosmobio.co.jp
Western blotting
SDS-sample buffer: 50 mM Tris/HCl, pH6.8, containing 2% SDS, (100 mM DTT), 10% glycerol and BPB
PBST: PBS containing 0.1% Tween 20
Blocking buffer: PBS containing 0.1% Tween 20 and 5% skim milk
Sample Preparation (for HeLa or NIH3T3 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 and collect 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.
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 4C. (overnight incubation is esssntial)
(13) Incubate the membrane with primary antibody diluted in Blocking buffer (1:500-1:1000) for 1 h at room temperature or
overnight at 4C. 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 (GE Healthcare NA9310V-1ML).
(15) Wash the membrane 3 times each for 5 min with PBST.
(16) Detect signals using an appropriate luminescent reagent.
*Clearer results can be obtained by using 'Can Get Signal (cat# TYB-NKB-101T)' during incubation with primary and secondary
antibodies, according to the manufacture's instructions.
For research use only. Not for clinical diagnosis.

Manufactured by BioAcademia,Inc.
TOYO 2CHOME, KOTO-KU, TOKYO, 135-0016, JAPAN

URL: http://www.cosmobio.co.jp
e-mail: [email protected]
[Outside Japan] Phone : +81-3-5632-9617
[] Phone : +81-3-5632-9610
FAX : +81-3-5632-9618
FAX : +81-3-5632-9619

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MONOCLONAL ANTIBODY

For research use only. Not for clinical diagnosis.

Catalog No. BAM-73-505-EX

Anti-ATF6 antibody, (37-1)

WB, Immunoprecipitation (IP) (less efficient than clone1-7)

Optimal dilutions/concentrations should be determined by the end user.

Anti-ATF6 antibody, (37-1)

Anti-ATF6 antibody, (37-1)

For research use only. Not for clinical diagnosis.

TOYO 2CHOME, KOTO-KU, TOKYO, 135-0016, JAPAN

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