IRE1 alpha Antibody - BSA Free

Novus Biologicals | Catalog # NB100-2324

Novus Biologicals
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Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse, Rat

Applications

Validated:

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Knockdown Validated

Cited:

Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Knockdown Validated

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all IRE1 alpha Primary Antibodies »

Product Specifications

Immunogen

This IRE1 alpha antibody was raised against a synthetic peptide within the human IRE1 alpha protein (within residues 700-800). [Swiss-Prot #O75460]

Localization

Endoplasmic reticulum

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

110 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Description

NB100-2324 is useful for the detection of endogenous total IRE1 Alpha protein. For detecting phospho-IRE1 Alpha (Ser-724), we suggest NB100-2323 and it is recommended to normalize phospho-IRE1 alpha band intensity/immunoreactivity with total-IRE1 alpha using NB100-2324 or NB110-59971.

Scientific Data Images for IRE1 alpha Antibody - BSA Free

Immunohistochemistry: IRE1 alpha Antibody [NB100-2324]

Immunohistochemistry: IRE1 alpha Antibody [NB100-2324]

IRE1-alpha-Antibody-Immunohistochemistry-NB100-2324-img0010.jpg
Western Blot: IRE1 alpha Antibody [NB100-2324]

Western Blot: IRE1 alpha Antibody [NB100-2324]

Western Blot: IRE1 alpha Antibody [NB100-2324] - Total IRE1 Alpha protein in lysates from Min6 cells which were transfected with GFP-siRNA or Ire1-siRNA. Theoretical molecular weight: 110 kDa.
Western Blot: IRE1 alpha Antibody [NB100-2324]

Western Blot: IRE1 alpha Antibody [NB100-2324]

Western Blot: IRE1 alpha Antibody [NB100-2324] - ALOX15 knockout blocks alcohol-induced up-regulation of IRE1a in mouse liver. Image from verified customer review.
Immunocytochemistry/ Immunofluorescence: IRE1 alpha Antibody [NB100-2324]

Immunocytochemistry/ Immunofluorescence: IRE1 alpha Antibody [NB100-2324]

Immunocytochemistry/Immunofluorescence: IRE1 alpha Antibody [NB100-2324] - IRE-1 alpha antibody was tested in HeLa cells at 1:200 against Dylight 488 (Green). Alpha tubulin and nuclei were counterstained against Dylight 568 (Red) and DAPI (Blue).
Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324]

Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324]

Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324] - Analysis of a formalin fixed mouse colon tissue section using total IRE1 alpha antibody (NB100-2324) at 1:300. Note a strong cytoplasmic signal in various cells of colonic villi (columnar epithelial and goblet cells) and the muscular layer. A few cells, potentially with active UPR, showed nuclear positivity also while the signal was negligible in cells at base of crypts and lamina propria.
Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324]

Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324]

Immunohistochemistry-Paraffin: IRE1 alpha Antibody [NB100-2324] - Analysis of a formalin fixed mouse colon tissue section using total IRE1 alpha antibody (NB100-2324) at 1:300. Note a strong cytoplasmic signal in various cells of colonic villi (columnar epithelial and goblet cells) and the muscular layer. Some cells, most likely with active UPR, depicted nuclear positivity also while the signal was negligible in crypts cells and lamina propria.
IRE1 alpha Antibody

Western Blot: IRE1 alpha Antibody [NB100-2324] -

The inositol-requiring enzyme 1 alpha (IRE1 alpha )/ X-box-binding-protein 1 spliced isoform (XBP1s) arm of unfolded protein response (UPR) is induced by angiotensin II in VSMCs. (A–C) The rat aortic VSMCs infected with adenovirus encoding GRP78 or control GFP (100 moi) for 48 h were stimulated with 100 nM AngII (AII) for 1–6 h & immunoblotting was performed as indicated. (A) Representative blots from 4 independent experiments. (B) Signal intensity was used to calculate the expression ratio of XBP1s to GAPDH. (C) Signal intensity was used to calculate the IRE1 alpha Ser724 phosphorylation ratio to the total IRE1 alpha. The bars in the graphs show the mean ± SD from 4 independent experiments. * indicates p < 0.05. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32679678), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
IRE1 alpha Antibody

Western Blot: IRE1 alpha Antibody [NB100-2324] -

Abnormal Paneth cells show ER stress.(A, B) Representative transmission electron microscopy images of Paneth cells at the base of ileal crypts in (A) ICR & (B) SAMP1/YitFc mice. Scale bars indicate 2 μm. (C, D) Quantitative analysis of (C) granule number & (D) ER lumen diameter in Paneth cells (n = 3/each week for SAMP1/YitFc mice). For the measurements, three Paneth cells were randomly selected from each mouse. (E) SDS–PAGE Western blot analysis of ER stress markers, pIRE1 alpha, ATF4, cleaved-ATF6, & GRP78 in ileal crypts (n = 4/each group). Total-IRE1 alpha & HPRT1 was used as loading control. (F) Relative expression level of ER stress markers calculated from the band intensity. Error bars represent mean ± SEM. (C, D, F) Statistical significance was evaluated by t test in (C, D), & one-way ANOVA followed by Tukey’s post hoc test in (F). P < 0.05 was considered statistically significant. *P < 0.05, †P < 0.01, §P < 0.001. E, ER; G, granules; N, nucleus; n.s., not significant. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32345659), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
IRE1 alpha Antibody

Western Blot: IRE1 alpha Antibody [NB100-2324] -

Western Blot: IRE1 alpha Antibody [NB100-2324] - Zn (II)-curc induces endoplasmic reticulum (ER) stress in mutant p53H273-carrying cells. (a) Representative photomicrographs of ER-Red Fluorescence staining in U373 cells untreated (Mock) or treated with Zn (II)-curc (100 µg/mL) for 16 h (Original magnification: 40×). (b) Quantization of ER content in U373 cells from ER-Red Fluorescence-stained cells. Mean fluorescence intensity (MFI) of each individual cell was normalized to cell size & expressed as fold-change compared with untreated cells at the same time point. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (c) Western blot analysis of p53, BiP, total (tot) IRE1 alpha, phosphorylated (p) IRE1 alpha, & XBP1 spliced (s) protein levels evaluated in U373 & HT29 cells untreated or treated with Zn (II)-curc (100 µg/mL) for 24 h. (d) Densitometric analysis was performed using Image J software to calculate the ratio of the protein levels, as detected in (c), vs. beta -actin. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (e) Total mRNA was extracted from U373 & HT29 cells untreated or treated with Zn (II)-curc (100 µg/mL) for 24 h. Spliced (s) Xbp1 gene expression was assayed by the polymerase chain reaction (PCR) of reverse-transcribed cDNA. Densitometric analysis was performed using Image J software to calculate the Xbp1s/28S ratio. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (f) p53 gene expression was assayed by PCR as in (e). The p53/28S ratio is indicated. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32138264), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for IRE1 alpha Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:100 - 1:250

Immunohistochemistry

1:250 - 1:500

Immunohistochemistry-Paraffin

1:250 - 1:500

Knockout Validated

reported in scientific literature (PMID 31159306)

Western Blot

1:1000 - 1:2000
Application Notes
NB100-2324 is useful for the detection of endogenous total IRE1 Alpha protein. For detecting phospho-IRE1 Alpha (Ser-724), we suggest NB100-2323 and it is recommended to normalize phospho-IRE1 alpha band intensity/immunoreactivity with total-IRE1 alpha using NB100-2324 or NB110-59971. In Western blot a band at ~110 kDa is observed. In ICC/IF endoplasmic reticulum staining was observed in HeLa cells. The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reviewed Applications

Read 2 reviews rated 4.5 using NB100-2324 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.05% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at -20C. Avoid freeze-thaw cycles.

Background: IRE1 alpha

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) and upregulates ER molecular chaperones in order to cope with ER stress. UPR is initiated by three ER-localized protein sensors: PERK (PKR-like ER kinase), ATF (activating transcription factor 6), and IRE1 alpha (inositol-requiring enzyme 1 alpha) (1). IRE1 alpha is correlated with X-box binding protein (XBP1) as a potent UPR transcriptional activator (2).

Transcriptional activation of UPR-responsive genes are regulated by the ATF6 and IRE1-XBP1 pathways, which are often regulated by HIFs and contribute to cell survival under ER hypoxic stress (3). UPR signaling can a) inhibit protein translation to restore cell function, b) activate signaling to increase production of molecular chaperones for protein folding, and c) initiate ubiquitination signaling that leads to degradation of misfolded proteins in ER.

IRE1 alpha acts as the sensor of unfolded proteins in the ER. IRE1 alpha not only promotes cell survival but can initiate apoptosis when accumulation of unfolded proteins in the ER causes stress. IRE1 alpha is essential for viability under stress conditions that cause unfolded proteins to accumulate in the ER. IRE1 alpha is a transmembrane protein that has both serine-threonine kinase and endoribonuclease activities and has a theoretical molecular weight of 110 kDa. When detecting phospho-IRE1 alpha, it is recommended to normalize its band intensity with total IRE1 alpha.

References

1. Zheng, W., Xie, W., Yin, D., Luo, R., Liu, M., & Guo, F. (2019). ATG5 and ATG7 induced autophagy interplays with UPR via PERK signaling. Cell Commun Signal, 17(1), 42. doi:10.1186/s12964-019-0353-3

2. Cho, Y. M., Kim, D. H., Lee, K. H., Jeong, S. W., & Kwon, O. J. (2018). The IRE1alpha-XBP1s pathway promotes insulin-stimulated glucose uptake in adipocytes by increasing PPARgamma activity. Exp Mol Med, 50(8), 102. doi:10.1038/s12276-018-0131-0

3. Xia, Z., Wu, S., Wei, X., Liao, Y., Yi, P., Liu, Y.,... Liu, J. (2019). Hypoxic ER stress suppresses beta-catenin expression and promotes cooperation between the transcription factors XBP1 and HIF1alpha for cell survival. J Biol Chem, 294(37), 13811-13821. doi:10.1074/jbc.RA119.008353

Long Name

Serine/threonine-protein kinase/endoribonuclease IRE1

Alternate Names

ERN1, hIRE1p, IRE1, Ire1-alpha

Gene Symbol

ERN1

Additional IRE1 alpha Products

Product Documents for IRE1 alpha Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

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Product Specific Notices for IRE1 alpha Antibody - BSA Free

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.

Citations for IRE1 alpha Antibody - BSA Free

Customer Reviews for IRE1 alpha Antibody - BSA Free (2)

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  • IRE1 alpha Antibody
    Name: Anonymous
    Application: Immunocytochemistry
    Sample Tested: mouse vascular smooth muscle cell
    Species: Mouse
    Verified Customer | Posted 11/19/2018
    IRE1 alpha Antibody - BSA Free NB100-2324
  • IRE1 alpha Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Liver
    Species: Mouse
    Verified Customer | Posted 03/07/2018
    ALOX15 knockout blocks alcohol-induced up-regulation of IRE1a in mouse liver.
    IRE1 alpha Antibody - BSA Free NB100-2324

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Protocols

View specific protocols for IRE1 alpha Antibody - BSA Free (NB100-2324):

Immunocytochemistry Protocol

Culture cells to appropriate density on suitable glass coverslips in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 5-10 minutes.
2. Remove the formalin and add 0.5% Triton-X 100 in TBS to permeabilize the cells. Incubate for 5-10 minutes.
3. Remove the permeabilization buffer and add wash buffer (i.e. PBS or PBS with 0.1% Tween-20). Be sure to not let the specimen dry out. Gently wash three times for 10 minutes.
4. Alternatively, cells can be fixed with -20C methanol for 10 min at room temperature. Remove the methanol and rehydrate in PBS for 10 min before proceeding.
5. To block nonspecific antibody binding incubate in 10% normal goat serum for 1 hour at room temperature.
6. Add primary antibody at appropriate dilution and incubate at room temperature for 1 hour or at 4 degrees C overnight.
7. Remove primary antibody and replace with wash buffer. Gently wash three times for 10 minutes.
8. Add secondary antibody at the appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove antibody and replace with wash buffer. Gently wash three times for 10 minutes.
10. Nuclei can be staining with 4',6' diamino phenylindole (DAPI) at 0.1 ug/ml, or coverslips can be directly mounted in media containing DAPI.
11. Cells can now be viewed with a fluorescence microscope.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow proper laboratory procedures for the disposal of formalin.

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for IRE1 alpha Antibody - BSA Free

Showing  1 - 2 of 2 FAQs Showing All

  • Q: I have a customer who is interested in antibody NB100-2324 for use with rat samples. It is listed as working with rat and for Western blot applications. Can you tell me which rat tissues were tested in Western blot?

    A: Previous productions were reported to work in rat. We have not tested the current lot directly on any rat samples so I do not have data on what tissues, or lysates were effective. We will still guarantee rat as a reactive species, as it is stated on our datasheet.

  • Q: We hope to know which position the antibody NB 100-2324 recognize within the endogenous IRE1 alpha. Thanks. Is the region 700 to 800 within the luminal domain, or transmembrane or cytosolic domain? Is this region highly conserved between Human and mouse?

    A:

    It looks like the 700-800 region is cytoplasmic. The homology in this region is 93%, and this product has already been validated to work in both human and mouse species.

    https://www.uniprot.org/uniprot/O75460

  • Q: I have a customer who is interested in antibody NB100-2324 for use with rat samples. It is listed as working with rat and for Western blot applications. Can you tell me which rat tissues were tested in Western blot?

    A: Previous productions were reported to work in rat. We have not tested the current lot directly on any rat samples so I do not have data on what tissues, or lysates were effective. We will still guarantee rat as a reactive species, as it is stated on our datasheet.

  • Q: We hope to know which position the antibody NB 100-2324 recognize within the endogenous IRE1 alpha. Thanks. Is the region 700 to 800 within the luminal domain, or transmembrane or cytosolic domain? Is this region highly conserved between Human and mouse?

    A:

    It looks like the 700-800 region is cytoplasmic. The homology in this region is 93%, and this product has already been validated to work in both human and mouse species.

    https://www.uniprot.org/uniprot/O75460

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