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HEF-1 Antibody [2G9]

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEF1 antibody [2G9] normal breast tissue and ductal carcinoma

Catalogue Number: IQ296

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Target Antigen

HEF1

Primary Description

Mouse Anti-HEF1 [2G9]

Catalogue Number

IQ296

Quantity

50ul

Concentration

1mg/ml

Clone

2G9

Host

Mouse 

Isotype

IgG1 kappa

Immunogen

Fusion protein, corresponding to amino acids 82-398 of Human HEF1.

Myeloma/fusion partners

SP2/0

Specificity

Does not recognize p130 Cas. Not tested on Sin1. This antibody mostly detects HEF1 localized at the focal adhesion sites

Species Reactivity

Mouse, Rat, Human

Purification

Protein G

Format

Purified antibody containing PBS 0.1% sodium azide;

Applications

WB, IP, ICC/IF, IHC-Fr, Flow Cyt

Dilutions

Optimal antibody dilution should be determined by titration, however as a guideline try; 

WB: 1/2000 - 1/6000. Predicted molecular weight: 93 kDa.

IP: 1/1000.

ICC/IF: Use at an assay dependent dilution. PubMed: 19376971

IHC-Fr: Use at an assay dependent dilution. PubMed: 19464348

Flow Cyt: Use 1µg for 106 cells

References

Kondo S et al. Impact of the integrin signaling adaptor protein NEDD9 on prognosis and metastatic behavior of human lung cancer. Clin Cancer Res. 2012 Nov 15;18(22):6326-38. doi: 10.1158/1078-0432.CCR-11-2162. Epub 2012 Oct 4. WB ; Human PMID: 23037767 

Bradbury P et al. PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9. Biochim Biophys Acta. 2012 Feb;1823(2):290-7. doi: 10.1016/j.bbamcr.2011.10.011. Epub 2011 Oct 28. IP WB ; Human PMID: 22061964

NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration. Zhong J et al. PLoS One. 2012;7(4):e35058. doi: 10.1371/journal.pone.0035058. Epub 2012 Apr 11 PMID: 22509381

The aryl hydrocarbon receptor regulates focal adhesion sites through a non-genomic FAK/Src pathway. Tomkiewicz C et al. Oncogene. 2012 Jun 4. doi: 10.1038/onc.2012.197. [Epub ahead of print] PUBMED

Li Y et al. HEF1, a novel target of Wnt signaling, promotes colonic cell migration and cancer progression. Oncogene. 2011 Jun 9;30(23):2633-43. doi: 10.1038/onc.2010.632. Epub 2011 Feb 14. ICC/IF IHC WB ; Human, Mouse PMID: 21317929

Bradshaw LN et al. Estradiol stabilizes the 105-kDa phospho-form of the adhesion docking protein NEDD9 and suppresses NEDD9-dependent cell spreading in breast cancer cells. Biochim Biophys Acta. 2011 Feb;1813(2):340-5. doi: 10.1016/j.bbamcr.2010.11.018. Epub 2010 Dec 8. WB ; Human PMID: 21145356

Hivert V et al. Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation. Biochem Pharmacol. 2009 Oct 15;78(8):1017-25. doi: 10.1016/j.bcp.2009.06.005. Epub 2009 Jun 17. WB ; Human PMID: 19539609

Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity. Bui LC et al. Oncogene. 2009 Oct 15;28(41):3642-51. doi: 10.1038/onc.2009.224. Epub 2009 Aug 3.  ICC/IF WB ; Human PMID: 19648964

Natarajan M et al. HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells.Oncogene. 2006 Mar 16;25(12):1721-32. ICC/IF IP WB ; Human PMID: 16288224 

Bargon SD et al. The Cas family docking protein, HEF1, promotes the formation of neurite-like membrane extensions. Biochim Biophys Acta. 2005 Dec 15;1746(2):143-54. Epub 2005 Nov 14. ICC/IF WB ; Human, Rat PMID: 16344118

Proteolysis of the docking protein HEF1 and implications for focal adhesion dynamics. ONeill GM ( Molecular and Cellular Biology,Aug 2001) PMID:11438665

Database links

Entrez Gene: 4739 Human

Entrez Gene: 18003 Mouse

Entrez Gene: 291044 Rat

Omim: 602265 Human

SwissProt: Q14511 Human

SwissProt: O35177 Mouse

Unigene: 37982 Human

Research areas

Apoptosis; Cell Biology; FAK Family; Intracellular; signal transduction; Transcription;

Also known as

CAS L antibody;Cas like docking antibody;CASL antibody;Crk associated substrate related proteinantibody;dJ49G10.2 antibody;dJ761I2.1 antibody;Enhancer of filamentation 1 antibody;HEF 1 antibody;NEDD 9antibody;NEDD9 antibody;NEDD9 protein antibody;Neural cell expressed developmentally down regulated 9antibody;Neural precursor cell expressed developmentally down regulated 9 antibody;NY REN 12 antigenantibody;p105 antibody;Protein NEDD9 antibody


THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC USE.

Print the Datasheet for HEF-1 Antibody [2G9]

  • HEF1 is a multifunctional protein involved in integrin-based signaling that affects cell motility growth apoptosis and oncogenic transformation The Cas family of docking proteins have been the subject of intense research because of their role in cell motility growth apoptosis and oncogenic transformation These proteins are substrates of focal adhesion kinase (FAK) and the Src family of tyrosine kinases two active targets for drug development HEF1 protein production increases levels of mRNA transcripts that encode proteins associated with motility cell transformation and invasiveness including several metalloproteinases MLCK p160ROCK and ErbBi HEF1 overproduction also mediates apoptosis in epithelial-derived cell lines including MCF7 and HeLa cells Recent clinical studies at another institution have found that overexpression of BCAR1 (p130Cas) a related protein is associated with tamoxifen resistance This highlights the importance of studying the role of this family of proteins in cancer prognosis

Immunofluorescence protocol - Formaldehyde fixation

   1. Collect cells from T.c.unit and remove media from petri dish using suction.
   2. Wash with 1x PBS and remove.
   3. Incubate cells in pre-warm (37°C) Para-Formaldehyde for 12 minutes at room temperature on an orbital shaker.
   4. Remove PFA and incubate in 0.5% Triton X-IOO in 1x PBS for 5 minutes at room temperature.
   5. Prepare blocking reagent, this is also the antibody diluent.
   6. Wash cells 2x with 1x PBS at room temperature, for 4 minutes/wash on an orbital shaker.
   7. Block with 1 % NCS and 1x PBS for 30 minutes at room temperature.
   8. Prepare primary antibodies (50?l/coverslip) and moist staining chambers.
   9. Wash cells 2x with lx PBS at room temperature and air dry briefly.
  10. Incubate with primary antibody for 1 hr at room temperature in the dark in staining chambers. During this time prepare the secondary antibody.
  11. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
  12. Incubate with secondary antibody for 1 hour at room temperature in the dark in staining chambers.
  13. Wash cells 5x with 1x PBS.
  14. Mount in Dapi.

Solutions (prepare fresh the same day of staining).

    * 1x Phosphate buffered saline.
    * Blocking reagent: 1% NCS in 1x PBS (use fresh l0x PBS).
    * Fixation solution: 3.5% Para formaldehyde.

1.75g PFA in 20 ml d.H20 plus 5 drops 1M NaOH. Stir on a hot plate at 50-60°C until dissolved. Add 4 drops IN HCI and check pH indicator strip. PH should be 7.4. Complete volume with d.H20 to 25ml and add 25ml 2xPBS. Check pH before adding to cover slips.


Immunofluorescence protocol - Methanol/acetone fixation

   1. Collect cells from T.C.unit and remove media from petri dish using suction.
   2. Wash with 1x PBS and remove.
   3. Fix cells with cold methanol: acetone 1: 1 for 10 minutes on ice.
   4. Prepare blocking reagent, this is also the diluent for the antibodies.
   5. Remove fixative and wash cells 3x with Ix PBS at RT, for 4 minutes/wash on orbital shaker.
   6. Block with 1% NCS and Ix PBS for 30 minutes at RT.
   7. Prepare primary antibodies (50?l/coverslip) and moist staining chambers.
   8. Wash cells 2x with 1 x PBS at RT and air dry for approximately 7 minutes.
   9. Incubate with primary antibody for 1 hr at RT in the dark in staining chambers.  During this time prepare secondary antibody.
  10. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
  11. Incubate with secondary antibody for 1 hr at R T in the dark in staining chambers.
  12. Wash cells 5x with 1x PBS.
  13. Mount in Dapi.

Solutions (prepare fresh the same day of staining)

    * 1x Phosphate buffered saline.
    * Blocking reagent: 1% NCS in 1x PBS (use fresh 10x PBS).
    * Fixation solution: methanol:acetone 1: 1 ice cold.



Western Blotting Protocol

   1. Transfer gel to PDVF or nitrocellulose membrane
   2. Place membrane in plastic tray in blocking buffer for one hour with agitation
   3. Rinse in wash buffer
   4. Incubate in wash buffer plus primary antibody for one hour
   5. Wash 6 X 5 minutes with wash buffer
   6. Incubate in wash buffer plus secondary antibody for one hour
   7. Wash 6X 5 minutes with wash buffer
   8. Detect (e.g. ECL, Amersham according to manufacturers instructions)

Wash buffer
PBS + 0.1% Tween 20

Blocking buffer
Wash buffer + 5% dried milk powder

The concentration of antibodies used depends on each antibody, the amount of antigen and the detection method used. Generally, dilution is in the range of a few hundred times dilution to a few thousand times dilution, but usually has to be determined empirically.

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How do I place an order?
How long does delivery take?
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How do I place an order?

When placing an order we require a purchase order number, plus name and contact details of the purchaser, and the person who will be using the product. We will add VAT for every order received from an EU country, unless you are VAT exempt. For such VAT exempt customers we will need to receive a copy of the exemption certificate with the order.

Orders can be placed either by our website, via email or by mail. Please see our contact us page for more details

All orders are subject to availability. Prices of products do not include shipping, VAT or import duties, where these are applicable.
Price and other information provided are subject to change without notice. While every effort is made to keep information provided Up to date, ImmuQuest will not be liable if errors should occur in such information.

Formal acceptance of an order will take place when the goods are dispatched. If prices should be changed between the time of receipt of an order and dispatch, ImmuQuest will contact you in advance.


Ordering via the website

To order via the website you will need to find the product(s) you are looking for by either using our search, browsing our categories or using our full antibody catalogue.

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How long does delivery take?

Delivery usually takes no more than one week from reciept of an order.

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Do you guarantee your antibodies?

Yes. Should any product not perform as described in the product literature, it will be replaced or a full refund will be given. The customer must notify ImmuQuest within 30 days after the goods have been received to request a replacement, forwarding complete test data as requested by ImmuQuest.

If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

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Do you charge VAT?

If you are in the EU and you are no exempt from paying VAT then we must charge VAT (17.5%).

Customers that are exempt need to fax an exemption form showing their vat exemption number.

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You can still place an order without providing a purchase order number but we must recieve payment before your order is despatched.

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Non-website orders

Non-website orders can be placed directly with Immuquest at sales@immuquest.com or click on the "contact us" tab for address details

 

Product Guarantee

We are strongly committed to providing the best quality products, and the best service possible. In order to do this we depend on your feedback.

Should any product not perform as described in the product literature, it will be replaced or a full refund will be given. The customer must notify ImmuQuest within 30 days after the goods have been received to request a replacement, forwarding complete test data as requested by ImmuQuest.

If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

If you have any questions please contact us using the following details:
ImmuQuest Ltd
26 The Green
Seamer
North Yorkshire
TS9 5LP
UK

Tel: 01642 713533
Fax: 01642 713988
International Tel: +44 1642 713533
International Fax: +44 1642 713988

When placing an order ImmuQuest require a purchase order number, plus name and contact details of the purchaser, and the person who will be using the product. ImmuQuest will also need a VAT number for customers in the European Union.UK customers that are VAT exempt need to fax an exemption certificate.

Orders can be placed either by our website, via email or by mail. Please see our contact us page for more details. All orders are subject to availability. Prices of products do not include shipping, VAT or import duties where these are applicable.

Price and other information provided are subject to change without notice. While every effort is made to keep information provided Up to date, ImmuQuest will not be liable if errors should occur in such information. Formal acceptance of an order will take place when the goods are dispatched. If prices should be changed between the time of receipt of an order and dispatch, ImmuQuest will contact you in advance.

Research Areas
Apoptosis Cell Biology FAK Family Intracellular signal transduction Transcription
Research
Proteolysis of the docking protein HEF1 and implications for focal adhesion dynamics - ONeill GM The Cas family docking protein HEF1 promotes the formation of neurite-like membrane extensions - Sharmilla D Bargon et al The Hematopoietic Isoform of Cas-Hef1-Associated Signal Transducer Regulates Chemokine-Induced Inside-Out Signaling and T Cell Trafficking - Adam G Regelmann et al

Offline Orders

For offline orders please call +44 (0) 1642 713533 or click here to email us.

Delivery & returns

We use federal Express for UK & international shipments, andfor standard shipments the cost is £30 UK & £55 international. If the customer uses their account, we charge a £20 handling fee (UK & international)

We are strongly committed to providing the best quality products, and the best service possible.

Should any product not perform as described in the product literature, it will be replaced or a full refund will be given. The customer must notify ImmuQuest within 30 days after the goods have been received to request a replacement, forwarding complete test data as requested by ImmuQuest.

If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

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