Anti-IRF5 antibody [10T1] KO Tested (ab33478)

Product name

Anti-IRF5 antibody [10T1]
See all IRF5 primary antibodies
Description

Mouse monoclonal [10T1] to IRF5
Host species

Mouse
Specificity

ab33478 recognises IRF-5.
Tested applications

Suitable for: ICC/IF, ELISA, IP, WB, Flow Cyt, IHC-Pmore details
Species reactivity

Reacts with: Mouse, Human
Immunogen

Recombinant human IRF-5 (176-240 aa, isoform D) purified from E. coli. NCBI Accession No.: NP_001092099 (formally NP_002191).
Positive control
- Extracts of Ramos, THP-1 and A20 cells. HEK293

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.40
Preservative: 0.1% Sodium azide
Constituent: 99% PBS
Concentration information loading...
Purity

Protein G purified
Clonality

Monoclonal
Clone number

10T1
Myeloma

Sp2/0
Isotype

IgG1
Light chain type

kappa
Research areas

Compatible Secondaries
Conjugation kits
- PE / R-Phycoerythrin Conjugation Kit (ab102918)
- APC Conjugation Kit (ab201807)
Immunohistochemistry kits
- Mouse on Mouse Polymer IHC Kit (ab127055)
Isotype control
- Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)
- Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control (ab91353)
Positive Controls

Our Abpromise guarantee covers the use of ab33478 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
ICC/IF		1/100 - 1/200.
ELISA		Use at an assay dependent concentration.
IP		Use at an assay dependent concentration.
WB		1/500 - 1/1000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).
Flow Cyt		Use 1µg for 10⁶ cells. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
IHC-P		1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Involvement in disease

Genetic variations in IRF5 are associated with susceptibility to inflammatory bowel disease type 14 (IBD14) [MIM:612245]. IBD14 is a chronic, relapsing inflammation of the gastrointestinal tract with a complex etiology. It is subdivided into Crohn disease and ulcerative colitis phenotypes. Crohn disease may affect any part of the gastrointestinal tract from the mouth to the anus, but most frequently it involves the terminal ileum and colon. Bowel inflammation is transmural and discontinuous; it may contain granulomas or be associated with intestinal or perianal fistulas. In contrast, in ulcerative colitis, the inflammation is continuous and limited to rectal and colonic mucosal layers; fistulas and granulomas are not observed. Both diseases include extraintestinal inflammation of the skin, eyes, or joints.
Genetic variations in IRF5 are associated with susceptibility to systemic lupus erythematosus type 10 (SLEB10) [MIM:612251]. Systemic lupus erythematosus (SLE) is a chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system.
Genetic variations in IRF5 are a cause of susceptibility to rheumatoid arthritis (RA) [MIM:180300]. It is a systemic inflammatory disease with autoimmune features and a complex genetic component. It primarily affects the joints and is characterized by inflammatory changes in the synovial membranes and articular structures, widespread fibrinoid degeneration of the collagen fibers in mesenchymal tissues, and by atrophy and rarefaction of bony structures.
Sequence similarities

Belongs to the IRF family.
Contains 1 IRF tryptophan pentad repeat DNA-binding domain.
Cellular localization

Nucleus.
Target information above from: UniProt accession Q13568 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 3663 Human
- Entrez Gene: 27056 Mouse
- Omim: 607218 Human
- SwissProt: Q13568 Human
- SwissProt: P56477 Mouse
- Unigene: 521181 Human
- Unigene: 6479 Mouse
Alternative names
- Interferon regulatory factor 5 antibody
- Interferon regulatory factor 5 bone marrow variant antibody
- IRF 5 antibody
- IRF-5 antibody
- Irf5 antibody
- IRF5_HUMAN antibody
- SLEB10 antibody
see all

Western blot - Anti-IRF5 antibody [10T1] (ab33478)

All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/500 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : IRF5 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 58 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab33478 observed at 56 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab33478 was shown to specifically react with IRF5 in wild-type HAP1 cells as signal was lost in IRF5 knockout cells. Wild-type and IRF5 knockout samples were subjected to SDS-PAGE. Ab33478 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)

Immunofluorescence/Immunocytochemistry analysis of HEK293 cells (10⁵-10⁶) stained with Mouse monoclonal [10T1] to IRF5 (ab33478 1/100-1/200).
Western blot - Anti-IRF5 antibody [10T1] (ab33478)

All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/1000 dilution

Lane 1 : Ramos cell lysate
Lane 2 : THP-1 cell lysate
Lane 3 : A20 cell lysate
Lane 4 : NIH 3T3 cell lysate

Secondary
All lanes : goat anti-mouse secondary antibody conjugated to HRP

Developed using the ECL technique.

Predicted band size: 58 kDa
Observed band size: 58 kDa
Additional bands at: 26 kDa, 80 kDa. We are unsure as to the identity of these extra bands.

100-200µg of whole cell lysates were used for western blotting.
Flow Cytometry - Anti-IRF5 antibody [10T1] (ab33478)

Overlay histogram showing THP1 cells stained with ab33478 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab33478, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in THP1 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [10T1] (ab33478)

ab33478 at 1/50 dilution staining IRF5 in Human normal colon mucosa tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The section was incubated with the primary antibody for 2 hours at room temperature. Antigen retrieval was performed in 0.1M sodium citrate buffer and detected using Diaminobenzidine (DAB).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [10T1] (ab33478)

ab33478 at 1/50 dilution staining IRF5 in Human colon adenocarcinoma tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The section was incubated with the primary antibody for 2 hours at room temperature. Antigen retrieval was performed in 0.1M sodium citrate buffer and detected using Diaminobenzidine (DAB).

Flow cytometry protocols
Immunoprecipitation protocols
Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

This product has been referenced in:

Xiao Y et al. An oligodeoxynucleotide with AAAG repeats significantly attenuates burn-induced systemic inflammatory responses via inhibiting interferon regulatory factor 5 pathway. Mol Med 23:N/A (2017). Read more (PubMed: 28620671) »
Koelzer VH et al. Digital analysis and epigenetic regulation of the signature of rejection in colorectal cancer. Oncoimmunology 6:e1288330 (2017). Read more (PubMed: 28507795) »

See all 12 Publications for this product

Publishing research using ab33478? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Application

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

Sample

Human Tissue sections (Macrophages)

Antigen retrieval step

Heat mediated - Buffer/Enzyme Used: Ventana Ultra CC1

Permeabilization

Specification

Macrophages

Blocking step

OptiView H2O2 as blocking agent for 8 minute(s) · Concentration: 0.04% · Temperature: 36°C

Fixative

Formaldehyde

Abcam user community

Verified customer

Submitted Jan 25 2016

Application

Western blot

Loading amount

30 µg

Gel Running Conditions

Reduced Denaturing

Sample

Human Cell lysate - whole cell (macrophage)

Specification

macrophage

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 12 2014

Question

Phone call reporting problems in using anti-IRF5 antibody [10T1] (ab33478) in western blotting.

Abcam community

Verified customer

Asked on Apr 18 2012

Answer

Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, I have attachedour questionnaire so that we can gather further information regarding the samples tested and the protocol used.This information will also allow us to investigate this case internally and initiate additional testing where necessary.

As discussed over the phone, I have arranged for a replacement vial of ab33478 to be sent out to you. This should reach you tomorrow or Friday. The order number is xxxxxx (Purchase order number FOCR xxxxx). If you do not find that your results improve with this new lot please do let me know.

I look forward to receiving your reply.

Abcam Scientific Support

Answered on Apr 18 2012

Application

Immunohistochemistry (Frozen sections)

Sample

Mouse Tissue sections (Brain)

Specification

Brain

Fixative

Paraformaldehyde

Permeabilization

Yes - heat, Triton X-100

Blocking step

Sniper (BioCare) as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 25°C

Ms. Colleen Karlo

Verified customer

Submitted Oct 04 2011

Question

I have a few questions about your anti-IRF5 antibody, cat.#:ab33478. Q1: what is the suggested concentration for detection of IRF5 in human dendritic cells? Q2: In the web site, the band shown in lane 2 is the endogenous IRF5 or inducible IRF5 in THP-1 cells? If it is inducible, what is the inducer and the concentration? Q3: Is ab6728 is the cat.# for HRP antibody used to show the IRF5 band? Q4: Dose milk or BSA used in blocking buffer make any differences in showing IFR5 expression? I would greatly appreciate your promptly response.

Abcam community

Verified customer

Asked on Jul 17 2006

Answer

Thank you for your interest in our product, anti-IRF5 antibody(ab33478). I now have some more details which should answer your questions. 1. The anti-IRF5 Antibody (ab33478) was used at 1:1,000 dilution, and 30~50mg protein of cell lysates was used for detection in western blotting. 2. Bands shown in the data sheet are endogenous IRF5, they are not inducible protein. 3. A commercialised secondary antibody, goat anti-mouse Ig-HRP conjugated was used. This was not ab6728. 4. You can get same results for milk or BSA as blocking solution, So you may use anything. I hope that theses answers are helpful for you and please contact me again if you have any other questions. With thanks.

Abcam Scientific Support

Answered on Jul 19 2006

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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Anti-IRF5 antibody [10T1] (ab33478)

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References

This product has been referenced in:

Customer reviews and Q&As

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-IRF5 antibody [10T1]

Western blot abreview for Anti-IRF5 antibody [10T1]

Immunohistochemistry (Frozen sections) abreview for Anti-IRF5 antibody [10T1]