Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Hypoxia leads to EMT and ER-stress in CRC cells. B. C. Confluent growing SW480 (B) and HCT116 (C) cells were cultured under conditions of normoxia or hypoxia-like ...read more
Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - Analysis of ATF6 in mouse liver tissue using 3 ug/ml of ATF6 antibody and 0.25 ug/ml of GAPDH antibody. Lane A contains 20 ugs of whole mouse liver lysate, lane B ...read more
Immunocytochemistry/ Immunofluorescence: ATF6 Antibody (70B1413.1) [NBP1-40256] - Untreated HeLa cells were fixed in -20C methanol for 10 min, air dried and rehydrated in PBS at room temperature for 5 minutes. Cells ...read more
Immunohistochemistry-Paraffin: ATF6 Antibody (70B1413.1) [NBP1-40256] - Human placenta, followed by biotinylated horse anti-mouse IgG secondary antibody, alkaline phosphatase-streptavidin and chromogen. Dilution 10ug/ml
Flow Cytometry: ATF6 Antibody (70B1413.1) [NBP1-40256] - Intracellular flow cytometric staining of 1 x 10^6 MCF-7 cells using ATF6 antibody (dark blue). Isotype control shown in orange. An antibody concentration of 1 ...read more
Western Blot: ATF6 Antibody (70B1413.1) [NBP1-40256] - 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. ...read more
(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.
(9) We highly recommend the use of a maximum sensitivity ECL substrate (Femto sensitive) for efficient detection of this antibody in Western blot applications.
This monoclonal antibody was made against a partial protein containing amino acids 1-273 of human ATF6.
This ATF6 antibody detects both the full length and the cleaved/active protein.
Protein G purified
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ICC: See Thomas et al (2005) and Kikuchi et al (2006) for details. Immunohistochemistry (Frozen): See Zhu et al (2008) for details. Immunohistochemistry (Paraffin): See van Kollenburg et al (2006) for details. Immunoprecipitation: See Hong et al, 2004 for details. Use in Immunoblotting reported in scientific literature (PMID 28550308). Knockout validation (PMID: 31531232). In Western blot do not use milk in the diluent as it can inhibit the observed signal. This antibody is CyTOF ready.
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.
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.
FAQs for ATF6 Antibody (NBP1-40256). (Showing 1 - 5 of 5 FAQs).
We are looking for an antibody specific to ATF6 alpha that will not cross-react with ATF6 beta. Would you please help confirm if any of the following are ATF6 alpha specific antibodies: NBP1-40256, NBP1-77251, NBP1-76675 & NBP1-41439?
The antibodies NBP1-77251 and NBP1-76675 are specific to ATF6 alpha and should not cross-react with ATF6 beta.
I learn from your company web that you are providing atf6, xbp1, cbx4 antibodies against human for immunofluorescence. And from the provided pictures, it seems very good. But I am wondering if anyone use them for immunofluorescence in human cells in your memory or in published papers? And how about them? By the way, you are providing free sample size for the atf6, xbp1 antibodies, aren't you? Could you send me some for tests? One more question, do they work on endogenous proteins but not only on overexpressed proteins?
These products have been validated for use in IF on human: catalog numbers NBP1-40256, NB100-80861, and NBP1-83225. Yes these antibodies should work on the native protein/endogenous protein.
Is there a blocking peptide available for your ATF6 antibody (NBP1-40256)?
This antibody was made against a partial recombinant protein (not a short peptide). As such we do not have a blocking peptide for this antibody nor do we have a blocking protein. We do however have a positive control, ready to use ATF6 lysate NBP1-70778.
IS THIS ATF6 ANTIBODY FOR WESTERN BLOT FROM MOUSE? SO WE NEED THE ANTI-MOUSE SECONDARY?
NBP1-40256 (ATF6 Antibody/70B1413.1) can be used in WB with mouse tissue/cell line samples. We have many secondary antibodies that can potentially be used with NBP1-40256. However, since the primary antibody has an isotype of IgG1, it is better to use the WB-validated anti-mouse IgG1 secondary antibodies.
We are having trouble with the following antibody, we did not observe any signal for ATF6 (full length or cleaved), only a non-specific band at around 37 kDa. We've tested on human MRC-5 fibrablasts. Do you have any suggestions that may be helpful to us?
The nature of the protein target ATF6 is differently expressed as the unglycosylated and/or glycosylated forms, with the different levels of glycosylation (http://www.uniprot.org/uniprot/P18850#ptm_processing), in the different organs/tissues/cells under the different physiological/pathological conditions. This ATF6 Antibody/70B1413.1 is a mouse monoclonal antibody with a very narrow epitope (even though we don't map any epitopes for our antibodies, it is known that almost all monoclonal epitopes have the sizes less than 15 a.a.). In other words, NBP1-40256 can bind to the full-length ATF6 and the long fragment of the cleaved protein target (not both cleaved protein species, and see #3 below). In order to detect the full-length and both two cleaved fragments of ATF6, the immunogen has to extend before and after the cleavage site at a.a. 419/420, in both directions, and therefore the epitopes can present in both sides of the cleavage site. ATF6 has 1 transmembrane domain, 1 leucine-zipper, and three glycosylation sites that could affect the mobility in electrophoresis. As the rule of thumb, we use 110 Da as an average of one amino acid. Therefore the full-length ATF6 gives 73,700 Da (vs. 74,585 Da calculated by the exact sequence). Since it is cleaved at a.a. 419 of ATF6, two fragments will be 419 a.a. and 251 a.a. (670 - 419). Take each of them and times 110 Da individually, and these are the results: 46,090 Da and 27,610 Da (the estimated values), respectively. Thefore your ~37 kDa species might be the short protein species with glycosylations (please double check with #1 above). Finally, we would like to suggest two things: please include other human cell lines as the controls: "http://www.proteinatlas.org/ENSG00000118217-ATF6/antibody" shows the WB positive results derived from using two cell lines and one organ. These could be used as the positive control. Also, the glycosylation levels are likely to be different in the different cells/tissues/organs, and therefor the different sizes of ATF can be viewed. The second thing is to use a real positive control lysate, such as ATF6 cell lysate or ATF6 overexpression lysate ( http://www.novusbio.com/productsearch?keys=atf6&_=1456165537185&fq[category]=Lysates), for the specificity of NBP1-40256.
A Key to Fight Stress: ATF6 The protein ATF6 is a constitutively expressed transcription factor that is a key mediator of the unfolded protein response (UPR) that allows mammalian cells to maintain cellular homeostasis under conditions of environmental and physiological stress.... Read full blog post.
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