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Product : Antibody >> Apoptosis , Cleavage Specific , Nuclear & Cytoplasmic.. , Transcription Factors >> ATF6 (Full-length and Active/Cleaved Forms)
      
 

Monoclonal Antibody to ATF6 (Full-length and Active/Cleaved Forms) (Clone 70B1413.1)

 
Cat. No.DescriptionFormatUnitPrice(USD)QuantityBulk Quotation
IMG-273
ATF6 (Full-length and Active/Cleaved Forms)Purified
0.1 mg
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Catalog No: IMG-273
Imgenex is now a part of Novus Biologicals! All orders, billing and shipments will be received from Novus. Please contact Novus directly for any questions – novus@novusbio.com. To insure proper processing of you order, please insure that your purchase orders show Novus Biologicals as the vendor.
Contents: 100 ug in 200 ul PBS containing 0.05% BSA and 0.05% sodium azide. Sodium azide is highly toxic.
Isotype: Mouse IgG1, Kappa
Clone: 70B1413.1
Purification: Protein G Chromatography
Species: Grouper (Fish), Hamster, Human, Mouse, Rabbit, Rat
Host: Mouse
Application: ChIP: See Donati et al, 2006 and Renna et al, 2007 for details.
IF/ICC: See Thomas et al (2005) and Kikuchi et al (2006) for details.
IHC (frozen): See Zhu et al, 2008 for details.
IHC (paraffin): See van Kollenburg et al (2006) for details.
IP: See Hong et al, 2004 for details.
Western blot analysis: 1-5 ug/ml
Storage: Store at 4°C, stable for 6 months. For long-term storage, aliquot and store at -20°C.
Recommended
Positive Control:
See Cat No. 40210 [ATF6 transfected cell lysates (transfected & mock transfected)]
 

Background
ATF6 is a constitutively expressed, endoplasmic reticulum (ER) membrane-anchored transcription factor.  ATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to environmental and physiological stresses that target the ER (reviewed in Shen, 2005 & Prywes, 2005). The C-terminus of ATF6 is located in the ER lumen and its N-terminal DNA binding domain faces the cytosol. AFT6 plays a key role in the ER stress response by transmitting the ER stress signal across the ER membrane into the nucleus.  The induction of new gene expression by ATF6 is an important aspect of the ER stress response. In response to certain stress conditions, ATF6 translocates from the ER to the Golgi.  The 90 kDa full-length ATF6 is processed within the Golgi to its active 50 kDa form through sequential cleavage by site-1 and site-2 proteases (S1P and S2P). Proteolytic activation of ATF6 in the ER stress response is a mechanism to regulate membrane-bound factors, and is referred to as regulated intramembrane proteolysis. The N-terminal active ATF6 translocates to the nucleus where it binds to ER stress-response elements in ER stress-response genes (ERSRGs). ATF6 is a potent transcriptional activator of ERSRGs. The fully glycosylated form of ATF6, a 670 amino acid protein, exhibits an electrophoretic mobility of ~90 kDa in denaturing SDS-gels, in part because of the glycosylated modifications.  ATF6 has 3 consensus sites for N-linked glycosylation and exists constitutively as a glycosylated protein.  Differentially glycosylated ATF6 forms may result from mutations or experimental treatment.

Antigen
This monoclonal antibody was made against a partial protein containing amino acids 1-273 of human ATF6.

Application Notes
(1) The ATF6 (IMG-273) has been cited to recognize both full-length and cleaved forms of ATF6. Please refer to the references in the Product Citation list for more comprehensive information. (2) The ATF6 transfected cell lysate (40210) is recommended as a useful western blot positive control to run in parallel with your experimental samples.

(3) Active/cleaved forms of ATF6 are generated through proteolytic cleavage during ER stress. Different molecular weights have been described for cleaved forms. A sample protocol was first described in Luo and Lee (2002) wherein NIH3T3 cells were treated with the amino acid analogue azetidine (AzC), and full-length and cleaved forms ATF6 were detected with IMG-273. The results show that in addition to the major 90 kDa full length ATF6, protein bands over the range of 50-70 kDa were detectable following AzC treatment (Luo and Lee, 2002: Figure 4A, page 791).  Many protocols and other cleaved forms have been described since, please consult the literature for additional information.

(4) For immunofluorescence microscopy, cells were fixed in methanol at -20 degrees C (Thomas et al, 2005).  Thomas et al used immunofluorescence to identify ATF6 with IMG-273 in the nucleus.  

(5) The active/cleaved 50 kDa nuclear form of ATF6 using IMG-273 has been found to be strongly expressed in certain tumor cell lines derived from B cell lymphoma (DEL), primary effusion lymphoma [BC-3 (ATCC CRL-2277), PEL-SY, HBL-6], lymphoblastic leukemia (DS-1) and multiple myeloma (RPMI-8226, NCI-H929), (Jenner et al. 2003).  

(6) Cleaved 60 and 36 kDa ATF6 forms have also been described in the nucleus (Mao et al, 2007).  

(7) The ATF6 antibody is reported to be specific for ATF6a, recognizing ATF6a but not ATF6b (Bommiasamy et al, 2009).  

(8) In western blots, the binding pattern of ATF6 may vary. Researchers are encouraged to consult the body of literature citing the ATF6 IMG-273 antibody (see Product citation list) for additional information. General ATF6 literature is also helpful. For example, Yoshida (1998) show a multiple band pattern in HeLa in both untreated and stressed cells (Fig 11B). In this early landmark ATF6 publication, multiple bands were seen between the 66 and 116 kDa markers as well as one or more bands between the 45 and 66 kDa markers.

Genebank Info (Protein): NP_031374
Gene ID: 22926
SwissProt: P18850
 
ATF6 (Full-length and Active/Cleaved Forms) Antibody

Western blot analysis of ATF6 in NIH3T3 cell lysate using IMG-273 at 3 ug/ml. A band corresponding to full-length ATF6 was detected. We have not characterized the ~36 kDa observed band; it may be an ATF6 breakdown/cleavage product.

ATF6 (Full-length and Active/Cleaved Forms) Antibody IMG-273

Western blot analysis of ATF6. Lane 1, 293 cells transfected with full-length ATF6.* Lane 2, 293 cells transfected with partial length ATF6 (amino acids 1-373).* Lane 3, Untransfected 293 cells.  Western blots were probed with 4 ug/ml of the ATF6 monoclonal antibody (IMG-273), followed by an HRP-conjugated second step (20101) and visualized with PicoTect Western Blot Chemiluminescence Substrate (10087K). Film was exposed for 1 min.  The top arrow corresponds to the ~90 kDa form of ATF6 described as full-length in the literature. *The  human full-length and partial length ATF6  plasmids are described in Luo and Lee (2002). 

ATF6 (Full-length and Active/Cleaved Forms)

Western blot analysis of ATF6 in mouse liver tissue enriched using 10088K using 3 ug/ml of IMG-273 (anti-ATF6) and 0.25 ug/ml of IMG-5143A (anti-GAPDH loading control).  Lane A contains 20 ugs of whole mouse liver lysate, lane B contains 20 ugs of total ER fraction, and lane C contains 20 ugs of rough ER fraction. The ATF6* band may represent under glycosylated or cleaved/active ATF6.

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Reference
1. Groenendyk J and M Michalak. 2005. Endoplasmic reticulum quality control and apoptosis. Acta Biochimica Polonica 52:381-295.
2. Shen J and R Prywes. 2005. ER stress signaling by regulated proteolysis of ATF6. Methods 35:382-389.


Research purposes only. Not for diagnostic or in vivo use. This product is guaranteed to perform as indicated on the datasheet for one year from the date of purchase.


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