Overview

  • Product name
    Anti-STAT1 (phospho Y701) antibody
    See all STAT1 primary antibodies
  • Description
    Rabbit polyclonal to STAT1 (phospho Y701)
  • Host species
    Rabbit
  • Specificity
    ab30645 detects endogenous levels of STAT1 only when phosphorylated at Tyrosine 701.
  • Tested applications
    Suitable for: ICC, ELISA, IP, IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic phosphopeptide derived from human STAT1 around the phosphorylation site of Tyrosine 701.

  • Positive control
    • IHC: Breast carcinoma tissue. WB: MC7 cells treated with EGF.

Properties

Applications

Our Abpromise guarantee covers the use of ab30645 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 Use at an assay dependent concentration.
ELISA 1/10000.
IP Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
WB 1/500 - 1/1000. Detects a band of approximately 87 kDa (predicted molecular weight: 87 kDa).
ICC/IF Use a concentration of 1 - 5 µg/ml.

Target

  • Function
    Signal transducer and activator of transcription that mediates signaling by interferons (IFNs). Following type I IFN (IFN-alpha and IFN-beta) binding to cell surface receptors, Jak kinases (TYK2 and JAK1) are activated, leading to tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize, associate with ISGF3G/IRF-9 to form a complex termed ISGF3 transcription factor, that enters the nucleus. ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of interferon stimulated genes, which drive the cell in an antiviral state. In response to type II IFN (IFN-gamma), STAT1 is tyrosine- and serine-phosphorylated. It then forms a homodimer termed IFN-gamma-activated factor (GAF), migrates into the nucleus and binds to the IFN gamma activated sequence (GAS) to drive the expression of the target genes, inducing a cellular antiviral state.
  • Involvement in disease
    Note=STAT1 deficiency results in impaired immune response leading to severe mycobacterial and viral diseases. In the case of complete deficiency, patients can die of viral disease.
    Defects in STAT1 are a cause of mendelian susceptibility to mycobacterial disease (MSMD) [MIM:209950]; also known as familial disseminated atypical mycobacterial infection. This rare condition confers predisposition to illness caused by moderately virulent mycobacterial species, such as Bacillus Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria, and by the more virulent Mycobacterium tuberculosis. Other microorganisms rarely cause severe clinical disease in individuals with susceptibility to mycobacterial infections, with the exception of Salmonella which infects less than 50% of these individuals. The pathogenic mechanism underlying MSMD is the impairment of interferon-gamma mediated immunity whose severity determines the clinical outcome. Some patients die of overwhelming mycobacterial disease with lepromatous-like lesions in early childhood, whereas others develop, later in life, disseminated but curable infections with tuberculoid granulomas. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance.
  • Sequence similarities
    Belongs to the transcription factor STAT family.
    Contains 1 SH2 domain.
  • Post-translational
    modifications
    Phosphorylated on tyrosine and serine residues in response to IFN-alpha, IFN-gamma, PDGF and EGF. Phosphorylation on Tyr-701 (lacking in beta form) by JAK promotes dimerization and subsequent translocation to the nucleus. Phosphorylation on Ser-727 by several kinases including MAPK14, ERK1/2 and CAMKII on IFN-gamma stimulation, regulates STAT1 transcriptional activity. Phosphorylation on Ser-727 promotes sumoylation though increasing interaction with PIAS. Phosphorylation on Ser-727 by PKCdelta induces apoptosis in response to DNA-damaging agents.
    Sumoylated by SUMO1, SUMO2 and SUMO3. Sumoylation is enhanced by IFN-gamma-induced phosphorylation on Ser-727, and by interaction with PIAS proteins. Enhances the transactivation activity.
    ISGylated.
  • Cellular localization
    Cytoplasm. Nucleus. Translocated into the nucleus in response to IFN-gamma-induced tyrosine phosphorylation and dimerization.
  • Information by UniProt
  • Database links
  • Alternative names
    • Signal transducer and activator of transcription 1 91kD antibody
    • CANDF7 antibody
    • DKFZp686B04100 antibody
    • IMD31A antibody
    • IMD31B antibody
    • IMD31C antibody
    • ISGF 3 antibody
    • ISGF-3 antibody
    • OTTHUMP00000163552 antibody
    • OTTHUMP00000165046 antibody
    • OTTHUMP00000165047 antibody
    • OTTHUMP00000205845 antibody
    • Signal transducer and activator of transcription 1 91kDa antibody
    • Signal transducer and activator of transcription 1 antibody
    • Signal transducer and activator of transcription 1, 91kD antibody
    • Signal transducer and activator of transcription 1-alpha/beta antibody
    • STAT 1 antibody
    • Stat1 antibody
    • STAT1_HUMAN antibody
    • STAT91 antibody
    • Transcription factor ISGF 3 components p91 p84 antibody
    • Transcription factor ISGF-3 components p91/p84 antibody
    see all

Images

  • All lanes : Anti-STAT1 (phospho Y701) antibody (ab30645)

    Lane 1 : Untreated MCF7 lysate (5-30ug).
    Lane 2 : EGF treated MCF7 lysate (5-30ug).

    Predicted band size: 87 kDa
    Observed band size: 87 kDa
    Additional bands at: 47 kDa. We are unsure as to the identity of these extra bands.



    Suggested dilution: 1:500 - 1:1000
  • ICC/IF image of ab30645 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab30645, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Paraffin-embedded human breast carcinoma tissue stained for STAT1 (phospho Y701) using ab30645 at 1/590 dilution in immunohistochemical analysis. Tissue was incubated in the absence (left) or presence (right) of immunizing phospho-peptide.

  • ab30645 staining STAT1 (phospho Y701) in murine intestine tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

    Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at 23°C; antigen retrieval was by heat mediation with a citrate buffer. Samples were incubated with primary antibody (1/400 in Tris Buffer Saline) for 16 hours at 4°C. An AlexaFluor®488-conjugated goat anti-rabbit polyclonal IgG (1/200) was used as the secondary antibody.

References

This product has been referenced in:
  • Zhao L  et al. Long non-coding RNA HULC promotes UVB-induced injury by up-regulation of BNIP3 in keratinocytes. Biomed Pharmacother 104:672-678 (2018). WB ; Human . Read more (PubMed: 29803927) »
  • Gang X  et al. Treatment effect of CDKN1A on rheumatoid arthritis by mediating proliferation and invasion of fibroblast-like synoviocytes cells. Clin Exp Immunol N/A:N/A (2018). Read more (PubMed: 29920650) »
See all 11 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer

Thank you fory our message which has been forwarded to me as my colleague ##### is away from the office today.

I am sorry I am not able to trace any previous correspondence from the email address you have used, and so I hope I can provide adequate information for you.

The concentrations and amounts can vary for each antibody, so I can recommend to review individual online datasheets for information. I can confirm the follownig concentrations for you:

STAT1 (ab2415)
We sell 1 ml at 0.2mg/ml
https://www.abcam.com/index.html?datasheet=2415 (or use the following: https://www.abcam.com/index.html?datasheet=2415).

pSTAT2 (ab53132)
We sell 100 ug at 1mg/ml (100 ul)
https://www.abcam.com/index.html?datasheet=53132 (or use the following: https://www.abcam.com/index.html?datasheet=53132).

STAT2 (ab53149)
We sell 100 ug at 1mg/ml (100 ul)
https://www.abcam.com/index.html?datasheet=53149 (or use the following: https://www.abcam.com/index.html?datasheet=53149).

pSTAT1 (ab30645)
We sell 100 ug at 1mg/ml (100 ul)
https://www.abcam.com/index.html?datasheet=30645 (or use the following: https://www.abcam.com/index.html?datasheet=30645).

I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

Read More
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Intestines)
Loading amount
15 µg
Specification
Intestines
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Oct 04 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Intestine)
Specification
Intestine
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 08 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Intestine)
Specification
Intestine
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted May 30 2012

Answer

Thank you for your patience.


The antibody ab31369 detects both alpha and beta forms of STAT1 when it is not phosphorylated at Y701. We believe that your treatment is increasing the expression of total STAT1 as well as the phosphorylated form.

Ab30645 detects the phosphorylation of Y701 whether or not the protein is phosphorylated at S727.

Please let me know if you need any additional information or assistance.

Read More

Answer

Thank you for your emails and for your patience while I've looked into these questions.

I've heard back from the lab regarding the specificity of ab31369. The antibody should detect both the alpha and beta forms, thoughwe haven't specifically tested whether it reacts with the phosphorylated protein too. There might be some reactivity with the phosphoprotein, but we would expect it to be weaker than reactivity with the non-phosphorylated protein. Based on our conversation last week and the images you sent, it looks like the antibody is reacting quite strongly with the phosphoprotein. The lab might be running some tests to determine this, and I'll keep you posted on anything else that we find out. I'll also need to check whether the antibody could detect STAT1 cleaved at residue 694, as I don't have the full immunogen seequence in my notes.

Ab30645 is specific for STAT1 phosphorylated at Y701, and I would expect it to react with protein phosphorylated at both Y701 and S727. Some phospho-specific antibodies show weak cross-reactivity for other phospho-proteins, so it is possible that ab30645 would weakly cross-react with protein only phosphorylated at S727. I'm not sure that this has been specifically tested by the lab, but I'll check with them and get back to shortly with that information.

Regarding the images, based on molecular weights I would expect the higher band to be the alpha isoform phosphorylated at both Y701 and S727, while the lower bands would be either the beta isoform (phosphorylated or not) or alpha isoform only phosphorylated at Y701. Phosphorylations eachtend to add some weight to the protein. I think further studies might need to be done in order to know this for sure, but that seems to be the most likely explanation. There are too many possible post-translational statesat similar molecular weights in order to know for sure what each band represents based on band size.

For ab11909, I may not be understanding the question as intended, but the full-length inactive ATF6 protein is cleaved by proteases during ER stress, so I would expect this form of the protein if the cells are under this type of stress. The full-length ATF6 most likely isn't completely lost, butthe cleaved form could certainly be more abundant in the sample and the inactive ATF6 at undetectable levels. It is also possible for proteases in the samples to cleave the protein during protein isolation/sample preparation but this would generally result in several lower molecular weight bands on the Western blot. If the samples are not treated with enough protease inhibitors (or the right mix of protease inhibitors) or were not kept on ice during preparation, it is possible for this to occur. If the samples were treated correctly, and the cells were under ER stress, than I expectthat specific proteases just cleaved the protein to yield the active form. Please let me know if I misunderstood the question or if there is more information that you're seeking.

I hope this information will be useful, but please let me know if you have further questions or if there is anything else that we can do for you. I'll be back in touch once I have more information from the lab about the STAT1 antibodies.

Read More

Answer

This antibody has given good staining in IHC on paraffin embedded slides when heat induced antigen retrieval with citrate buffer pH6.0 has been used. Enzyme is not needed. Please find here below also the protocol details used for testing this antibody in IHC-P: Deparaffinization 1.Warm up slide for 60 minutes at 60℃ incubator. 2.Deparaffinize in Xylene for 10 minutes and repeat one more times. 3.Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85%alcohol for 5 minutes, in 75% alcohol for 5 minutes. 4.Dip into Distill Water for 5 minutes. 5.Dip into TBS buffer (50 mM Tris, 100 mM NaCl, pH 7.6) for for 5 minutes, andrepeat two times with changes of TBS buffer. Antigen Retrieval 6.Boil 500 - 2000 ml 10 mM citrate buffer (pH6.0) in a stainless steel pressurecooker. 7.Put the slide into a staining rack, then citrate buffer (ensuring the slidewell immersed in citrate buffer). 8.When the pressure indicator valve has risen after 3-4 minutes, incubate for 1minute. 9.Cool the slide naturally to room temperature. 10.Dip into distilled water, leave for 5 minutes, and repeat two times. 11.Dip the slide in TBS for 5 minutes and repeat two times. 12.Immerse slides in 3% H2O2 (infresh methanol) for 15 minutes at room temperature. 13.Wash with distilled water two times, 5 minutes each time. 14.Wash with TBS (pH 7.6) two times, 5 minutes each time. 15.Place the slide into Blocking Solution (3% BSA in TBS) for 30 minutes.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Human Cultured Cells (Ovarian, Breast)
Specification
Ovarian, Breast
Fixative
Formaldehyde
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Apr 02 2010

Application
Western blot
Sample
Human Cell lysate - whole cell (Human B-cell lymphomas)
Loading amount
20 µg
Specification
Human B-cell lymphomas
Gel Running Conditions
Reduced Denaturing (8% Acrylamide gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Prof. Paul Townsend

Verified customer

Submitted Mar 31 2010

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