Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)

Rabbit Recombinant Monoclonal IRE1 phospho S724 antibody. Suitable for Dot, ICC/IF, IHC-P, WB and reacts with Synthetic peptide, Human, Mouse samples.

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Images

Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (AB324051), expandable thumbnail

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	Dot	ICC/IF	IHC-P	WB
Human	Expected	Tested	Tested	Tested
Mouse	Expected	Not recommended	Tested	Tested
Synthetic peptide	Tested	Not recommended	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info 1/1000	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Human, Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/5000	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Mouse	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000	Notes -
Species Mouse	Dilution info 1/1000	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide	Dilution info -	Notes -

Target data

See full target information ERN1 phospho S724

Function

Serine/threonine-protein kinase and endoribonuclease that acts as a key sensor for the endoplasmic reticulum unfolded protein response (UPR) (PubMed:11175748, PubMed:11779464, PubMed:12637535, PubMed:21317875, PubMed:28128204, PubMed:30118681, PubMed:9637683). In unstressed cells, the endoplasmic reticulum luminal domain is maintained in its inactive monomeric state by binding to the endoplasmic reticulum chaperone HSPA5/BiP (PubMed:21317875). Accumulation of misfolded proteins in the endoplasmic reticulum causes release of HSPA5/BiP, allowing the luminal domain to homodimerize, promoting autophosphorylation of the kinase domain and subsequent activation of the endoribonuclease activity (PubMed:21317875). The endoribonuclease activity is specific for XBP1 mRNA and excises 26 nucleotides from XBP1 mRNA (PubMed:11779464, PubMed:21317875, PubMed:24508390). The resulting spliced transcript of XBP1 encodes a transcriptional activator protein that up-regulates expression of UPR target genes (PubMed:11779464, PubMed:21317875, PubMed:24508390). Acts as an upstream signal for ER stress-induced GORASP2-mediated unconventional (ER/Golgi-independent) trafficking of CFTR to cell membrane by modulating the expression and localization of SEC16A (PubMed:21884936, PubMed:28067262).

Alternative names

IRE1, ERN1, Serine/threonine-protein kinase/endoribonuclease IRE1, Endoplasmic reticulum-to-nucleus signaling 1, Inositol-requiring protein 1, Ire1-alpha, hIRE1p, IRE1a

Rabbit Recombinant Monoclonal IRE1 phospho S724 antibody. Suitable for Dot, ICC/IF, IHC-P, WB and reacts with Synthetic peptide, Human, Mouse samples.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogens: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR26009-269
Purification technique: Affinity purification Protein A
Specificity: Unsuitable for mouse ICC
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

9 product images

Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Western blot staining using rabbit Anti-IRE1 (phospho S724) antibody
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Extra bands around 37 kDa were observed.
Exposure time: Lane 1, 4: 3 minutes Lane 2, 3: 81 seconds
All lanes: Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051) at 1/1000 dilution
Lane 1: Human cerebellum tissue lysate at 20 µg
Lane 2: Mouse cerebellum tissue lysate at 20 µg
Lane 3: SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg
Lane 4: Mouse brain tissue lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 110 kDa
Dot Blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Dot Blot staining using rabbit Anti-IRE1 (phospho S724) antibody
Dot blot analysis of IRE1 (phospho S724) using ab324051 at 1:1000 (0.528 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1:100,000 dilution.
Lane1: IRE1 phospho S724 peptide a

Lane2: IRE1 phospho S724 peptide b

Lane3: IRE1 non-phospho peptide

Lane4: IRE1 phospho S724+S726 peptide

Lane5: IRE1 phospho S726 peptide
Exposure time: 3 minutes.
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
This antibody does cross-activity with IRE1 (phospho S726) at higher concentrations by dot blot.
All lanes: Dot Blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051) at 1/1000 dilution
Lane 1: IRE1 phospho S724 peptide a
Lane 2: IRE1 phospho S724 peptide b
Lane 3: IRE1 non-phospho peptide
Lane 4: IRE1 phospho S724+S726 peptide
Lane 5: IRE1 phospho S726 peptide
Secondary
All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Exposure time: 3min
Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Western blot staining using rabbit Anti-IRE1 (phospho S724) antibody
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Phosphorylation of IRE1 at S724 can be induced by DTT treatment (PMID: 31453810).
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
All lanes: Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051) at 1/1000 dilution
Lane 1: Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg
Lane 2: HeLa treated with 10mM DTT for 1 hour whole cell lysate (untreated membrane) at 20 µg
Lane 3: Untreated HeLa whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 4: HeLa treated with 10mM DTT for 1 hour whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 110 kDa, 36 kDa
Exposure time: 3min
Immunocytochemistry/ Immunofluorescence - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-IRE1 (phospho S724) antibody
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling IRE1 (phospho S724) with ab324051 at 1/5000 (0.106 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 1000 2ug/ml dilution (Green).
Confocal image showing increased nuclear staining in HeLa cells (shown in green) treated with 10 mM for 1 hour, and the signal is decreased after Lambda Protein Phosphatase treatment at 30℃ for 2 hours. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 1000 2ug/ml dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-IRE1 (phospho S724) antibody
Immunohistochemical analysis of paraffin-embedded (A) Untreated HeLa (human cervical adenocarcinoma epithelial cell) (B) HeLa treated with 10mM DTT for 1 hour (C) Untreated HeLa treated with alkaline phosphatase (D) HeLa treated with 10mM DTT for 1 hour, then treated with alkaline phosphatase tissue labeling IRE1 (phospho S724) with ab324051 at 1/2000 (0.264 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Positive staining in (A) untreated HeLa cell pellets, staining was enhanced in (B) HeLa cell pellets treated with 10mM DTT for 1 hour. No staining in all cell pellets after treatment with alkaline phosphatase (C and D).

The section was incubated with ab324051 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-IRE1 (phospho S724) antibody
Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling IRE1 (phospho S724) with ab324051 at 1/2000 (0.264 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Postive staining on mouse spleen without alkaline phosphatase treatment (A). No signal was detected when tissues were treated with alkaline phosphatase (B).

The section was incubated with ab324051 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-IRE1 (phospho S724) antibody
Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling IRE1 (phospho S724) with ab324051 at 1/2000 (0.264 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Postive staining on human tonsil without alkaline phosphatase treatment (A). No signal was detected when tissues were treated with alkaline phosphatase (B).

The section was incubated with ab324051 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-IRE1 (phospho S724) antibody
Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling IRE1 (phospho S724) with ab324051 at 1/2000 (0.264 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Postive staining on human endometrium without alkaline phosphatase treatment (A). No signal was detected when tissues were treated with alkaline phosphatase (B).

The section was incubated with ab324051 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051)
IRE1 (phospho S724) Western blot staining using rabbit Anti-IRE1 (phospho S724) antibody
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The band above 250 kDa may be caused by aggregation.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
Exposure time: Lane 1, 2, 5, 6: 26 seconds Lane 3, 4, 7, 8: 3 minutes
All lanes: Western blot - Anti-IRE1 (phospho S724) antibody [EPR26009-269] (ab324051) at 1/1000 dilution
Lane 1: Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate (untreated membrane) at 20 µg
Lane 2: Neuro-2a treated with 10mM DTT for 1 hour whole cell lysate (untreated membrane) at 20 µg
Lane 3: Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage)whole cell lysate (untreated membrane) at 20 µg
Lane 4: RAW 264.7 treated with 10mM DTT for 1 hour whole cell lysate (untreated membrane) at 20 µg
Lane 5: Untreated Neuro-2a whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 6: Neuro-2a treated with 10mM DTT for 1 hour whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 7: Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage)whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 8: RAW 264.7 treated with 10mM DTT for 1 hour whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 110 kDa, 36 kDa