Overview

  • Product name
    Anti-STAT6 antibody [YE361]
    See all STAT6 primary antibodies
  • Description
    Rabbit monoclonal [YE361] to STAT6
  • Host species
    Rabbit
  • Specificity
    The antibody does not cross-react with other Stat family members.
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human STAT6 aa 800 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: P42226

  • Positive control
    • WB: Raji, NIH/3T3 and RAW 264.7 cell lysates. IHC-P: Human skin carcinoma, kidney, transitional cell carcinoma of bladder, glioma and solitary fibrous tumor tissues; Mouse kidney tissue. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. IP: NIH/3T3 cell lysate.
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32520 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
WB 1/1000 - 1/2000. Detects a band of approximately 100 kDa (predicted molecular weight: 94 kDa).
IHC-P 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF 1/100.
IP 1/20 - 1/80.
Flow Cyt Use at an assay dependent concentration.

Target

  • Function
    Carries out a dual function: signal transduction and activation of transcription. Involved in interleukin-4 signalling.
  • Sequence similarities
    Belongs to the transcription factor STAT family.
    Contains 1 SH2 domain.
  • Post-translational
    modifications
    Tyrosine phosphorylated following stimulation by IL-4 and IL-3.
  • Cellular localization
    Cytoplasm. Nucleus. Translocated into the nucleus in response to phosphorylation.
  • Information by UniProt
  • Database links
  • Alternative names
    • 12S1644 antibody
    • D12S1644 antibody
    • IL 4 STAT antibody
    • IL-4 Stat antibody
    • IL4 STAT antibody
    • Interleukin 4 Induced antibody
    • Interleukin 4 Induced Transcription Factor IL4 STAT antibody
    • Signal transducer and activator of transcription 6 antibody
    • Signal Transducer And Activator Of Transcription 6 Interleukin 4 Induced antibody
    • Signal Transducer And Activator Of Transcription 6 Nirs Variant 1 antibody
    • Signal transducer and activator of transcription 6, interleukin 4 induced antibody
    • STAT 6 antibody
    • STAT interleukin4 induced antibody
    • STAT, interleukin4 induced antibody
    • Stat6 antibody
    • STAT6_HUMAN antibody
    • STAT6B antibody
    • STAT6C antibody
    • Transcription factor IL 4 STAT antibody
    see all

Images

  • All lanes : Anti-STAT6 antibody [YE361] (ab32520) at 1/1000 dilution (purified)

    Lane 1 : NIH/3T3 (mouse embyro fibroblast cell line) cell lysate
    Lane 2 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

    Predicted band size: 94 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemical staining of paraffin embedded human kidney with purified ab32520 at a working dilution of 1/50. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
  • Immunofluorescence staining of HeLa (human epithelial cell line from cervix adenocarcinoma) cells with purified ab32520 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab32520 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

  • Flow Cytometry analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling STAT6 with purified ab32520 at 1/30 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1:2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

  • Anti-STAT6 antibody [YE361] (ab32520) at 1/20000 dilution (purified) + Raji (human Burkitt's lymphoma cell line) cell lysate at 10 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

    Predicted band size: 94 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skin carcinoma tissue labelling STAT6 with unpurified ab32520 at 1/100.

  • ab32520 (purified) at 1/20 immunoprecipitating STAT6 in 10 μg NIH/3T3 (mouse embyro fibroblast cell line; Lanes 1 and 2, observed at 100 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP (ab131366) was used as the secondary antibody (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

  • Immunocytochemistry/Immunofluorescence analyis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling STAT6 with unpurified ab32520 at 1/100.

  • Anti-STAT6 antibody [YE361] (ab32520) at 1/1000 dilution (unpurified) + NIH/3T3 (mouse embyro fibroblast cell line) cell lysate

    Predicted band size: 94 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma of bladder tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human solitary fibrous tumor tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

References

This product has been referenced in:
  • Zhang X  et al. Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype. J Cell Mol Med 22:409-416 (2018). WB ; Mouse . Read more (PubMed: 28944992) »
  • Zhao J  et al. Sphingosine-1-phosphate receptor-2 facilitates pulmonary fibrosis through potentiating IL-13 pathway in macrophages. PLoS One 13:e0197604 (2018). Read more (PubMed: 29782549) »
See all 11 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Sample
Mouse Tissue lysate - whole (LUNG)
Gel Running Conditions
Reduced Denaturing (7.5)
Loading amount
30 µg
Treatment
Oxidant 24hr
Specification
LUNG
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Dec 11 2015

Answer

Thank you for contacting us.



The antibody is from tissue culture supernatant.



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Answer

The Jurkat lysate will serve as a positive control for STAT6, in addition to IRF7. Although ab32520 has not been tested on Jurkat, other STAT6 antibodies have been, and the results demonstrate that Jurkat expresses this protein. For an example western blot of Jurkat stained for STAT6, please see the datasheet for ab125303: https://www.abcam.com/stat6-antibody-ab125303.html.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1180962.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

Please find enclosed the answers to your questions. I hope these answers will help in finding a solution to the none working antibody. Thank you in advance for looking into it. If you need high resolution pictures, please let me know.

Kind regards,

Order Details

Antibody code: ab32520

Lot number: GR17723

General Information

Antibody storage conditions (temperature/reconstitution etc) Storage at -20°C

Description of the problem (high background, low signal, non-specific satining etc.) No signal

Sample (Species/Tissue/Cell Type/Cell Line etc.) Prostate (TUR), Mammatissue including skin, Lymfnode, all Human

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.) Formaline 4%, duration 12 - 24h

Antigen retrieval (Enzymatic method, Heat mediated technique etc.) HIER. two methods used [1] CC1 Tris/borate/EDTA, pH 8.4 and [2] CC2 Citrate buffer equivalent, pH 6 (both ready to use buffer for Benchmark XT Ventana Medical Systems)

Permeabilization step: none

Blocking conditions (Buffer/time period, Blocking agent etc.) automatical via detection kit, 3% H2O2 to inhibit endogenous peroxydase, t= 4 min, protein block directly with incubatie secundairy Ab with goat serum and casein t = 8 min.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step), Rabbit anti STAT6 (ab32520), ABCAM, used dilution from 1:50 till 1:500 incubated 32 min at 37C and once 1:50 incubated 90 min at 37C.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Ultraview Multimer Ig, Ready to use mixture of Goat anti Mouse IgG, Goat anti mouse IgM, Goat anti Rabbit IgG, simultaneus with protein block. t=8 min.

Detection method Ultraview tm DAB detection kit (ventana medical systems)

Positive and negative controls used (please specify): above mentioned tissue are used as a positive control for optimalisation steps.

Optimization attempts (problem solving) see What steps have you altered

How many times have you tried the IHC? 5x

Have you run a No Primary control? No, not neccesary, while their is no signal. Only run this step when obtaining huge background.

Do you obtain the same results every time? Yes, no signal

What steps have you altered? changing antibody dilution, incubation time, antigen retrieval method, time of antigen retrieval and tissue.

Additional Notes: Optimization for automatic IHC system Benchmark XT (Ventana Medical Systems)

We would appreciate if you are also able to provide and image which woudl help us to assess the results

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Answer

Thank you for taking the time to complete our questionnaire and contact us. The details you have kindly provided will provide us with vital information for our monitoring of product quality.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details.

I can recommend you may like to consider trying the following options:

1. The time for antigen retrieval can sometimes require some optimization. For example I can recommend to try , 5, 10 and 20 minutes if this has not already been done.

2. We recommend not to mix blocking agents in one experiment. I can recommend to try serum only (no casein).

3. I can suggest to try overnight incubation at 4oC. This sometimes provides more specific and efficient staining.

4. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch.

I would also like to reassure you that this antibody is tested and covered by our 6 month guarantee for IHC-P and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:
Lot number:
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, low signal, non-specific satining etc.)
Sample (Species/Tissue/Cell Type/Cell Line etc.)
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the IHC?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
What steps have you altered?
Additional Notes
We would appreciate if you are also able to provide and image which woudl help us to assess the results

Read More

Answer

Thank you for contacting Abcam regarding ab32520.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

Thank you for contacting us regarding ab32520. I'm sorry to hear you are experiencing problems with this antibody which has been tested in IHC-P and should work in lung tissue of mouse and human. Could you please confirm the species of your samples? If it is not from the above species then the problem may be that the antibody does not recognise the species you are working with. I would like to recommend trying the following steps to improve your signal: 1)reduce the fixation time as overfixed tissue shows modified antigens which cannot be recognized by the antibody 2) vary the antigen retrieval time as too little or too much can also modify the antigen such that it is not optimally recognized by the antibody 3) use PBS containing 0.3% triton to dilute the blocking buffer and antibodies in 4) check that the tissue is washed in PBS a lot before adding the antibody, as the remaining fixative can fix the antibody and prevent its binding 5) check that the secondary antibody works with other primary antibodies, as well as your ABC kit. As you have tried this staining once I would first recommend to run this again as it may be that there was a problem in one of the steps in your first attempt. I can also please make sure that endogenous peroxidases are blocked? I hope this information helps, please do not hesitate to contact us if you need any more advice or information or still experience a problem following the modifications advised. I can offer you a refund or replacement vial if the antibody was purchased in the last 90 days,

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Question
Answer

Thank you for your reply. For ab32520, HIER using Citrate buffer pH 6 was used as the pretreatment on FFPE tissue. You may find a protocol for this at the following link: https://www.abcam.com/assets/pdf/protocols/IHC-paraffin%20protocol%20(IHC-P).pdf I hope this information helps. Please do not hesitate to contact us if you need anything further.

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