rev:      January 26, 2005

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  ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of researchd Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.

ANTI-Endothelin Receptor A & B (see also sample IP and histo protocol below)

-see also new rabbit polyclonals below

cat# RDI-ET1RECPabm        $438.00/50ug

        RDI-ET1RECPabm-1    $750.00/150ug

Background: Endothelin-1 (ET-1) is a potent vasoconstrictor that is secreted by vascular endothelial cells. ET-1, ET-2, ET-3, and endothelin b are members of a family of related peptides. All are 21 amino acid peptides with two disulfide bridges. ET-1 and ET-2 differ by only two residues, while ET-3 differs from both by six residues. ET-1 and ET-2 are highly effective on vascular smooth muscle, while the effects of ET-3 are minimal. ET-1 and its precursor, big ET-1, are the primary forms present in the circulation. Circulating ET-1 plays a pathologic role in the etiology of hypertension and antagonists are utilized in the treatment of this disease. The ET-1 receptor is a seven-transmembrane domain receptor of the G-protein-coupled family that signals via transient increases in intracellular Ca 2+. It is primarily expressed in lung and heart and is detected at lower levels in liver, brain, muscle, and kidney. ET-1 receptor interacts with ET-1, ET-2, and ET-3. However, it has a 2-3 fold higher affinity for ET-1 than ET-2 and the affinity for ET-3 is more than 100 fold lower. Thus, ligation of the ET-1 receptor by ET-1 induces increased intracellular Ca 2+ that signals to promote vasoconstriction.

Isotype: mIgG1

package: 50ug (200ul) or 150ug (600ul) (at 0.25mg/ml in 50% glycerol, PBS + 1.5mM NaN3 & 1mg/ml BSA)

antigen: a peptide protein fragment corresponding to amino acids 230-352 from rat endothelin 1 receptor

USe: Suitable for western blot on human, rat and mouse under using approx 0.5ug/ml ab on total cell of rat testis lysate.(2-3 Hour RT or over night at 4 DEg C with chemiluminescence detection). Major band =MW 50kDa. Immunoreactivity also shown with Immunofluorescence

Storage: Store at -20 DEG C upon receipt. . Recommend aliquoting. Avoid frequent warming cycles.

ref: -Lin, H.Y., et al. 1991 Proc. Natl. Acad. Sci. USA 88:3185- 3189

      -Masuda, Y., et al. 1989 FEBS Letters 257:208-210

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

DATA SHEET: Sheep anti-Endothelin B Receptor

Catalog#: RDI-ENDTHLBRabS     Price: $440.00/250ug vial           

Batch: see sheet with shipment

Package Size: packaged at 1mg/ml concnetration in 0.15M PBS, pH 7.5 (no preservatives)

Antigen: C-terminal peptide of rat endothelin B receptor conjugated to KLH.

                   sequence: Q T F E E K Q S L E E K Q               

Species: Sheep IgG purified by Protein G chromatography

USE: -Western Blot: aprox 20ug/ml (identifies 39Kd band)

          -Immunohistochemistry 20ug/ml (parrafin sections).

          -immunoprecipitation

           Note: These antibodies bind epitopes on the cytoplasmic side of the receptors,

                     thus the cell membrane must be permeable to antibodies. They will not block binding

                    of endothelin to the receptors.

Specificity: Tested with endothelin B receptor transfected COS cells. reacts with human, mouse and sheep.

Storage: Store at 4 DEG C (short term,) -20 DEG C long term. Avoid frequent freeze thaw cycles.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics

Also available: Anti-Endothelin A Receptor cat#RDI-ENDTHLARabS     (same price)

anti-endothelin antibodies (see alphabetical link)

For In Vitro Research Use Only-Not For Use In Or On Humans or Animals

Catalog#: RDI-ENDTHLARabS     Price: $440.00/250ug vial   

Batch: see sheet with shipment

Package Size: packaged at   mg/ml concentration in 0.15M PBS, pH 7.5 (no preservatives)
                     (VIAL CONTAINS 66UL/250UG).

Antigen: C-terminal peptide of rat endothelin A receptor conjugated to KLH.

               Sequence: 410-422     Q E Q N H N T E R S S H K

Species: Sheep IgG purified by Protein G chromatography

USE: -Western Blot: aprox 20ug/ml    (identifies 39Kd band)

-Immunohistochemistry 20ug/ml (parrafin sections).

-immunoprecipitation

-Elisa

Note: These antibodies bind epitopes on the cytoplasmic side of the receptors, thus the cell membrane must be permeable to antibodies. They will not block binding of endothelin to the receptors.

Specificity: Tested with endothelin A receptor transfected COS cells. Also reacts with human, mouse, rat and sheep.

Storage: Store at 4 DEG C (short term,) -20 DEG C long term. Avoid frequent freeze thaw cycles.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics

TECHNICAL BULLETIN

For Use With Anti-Endothelin A & Anti-Endothelin B Polyclonal  Antibodies.

Techniques For Immunocytochemical Staining & Immunoprecipitation

N.B.: These PAbs recognize epitopes on the cytoplasmic side of the receptors. Thus, to efective use these antibodies, the cell membrane must be permeable to antibodies. These antibodies, therefore, are of significant benefit since they will not block the binding of endothelins to the receptors. (try with and without  0.1% saponin for various tissues)

IMMUNOCYTOCHEMICAL STAINING for ET-A and ET-B receptors

Fixed paraffin-embedded sections (4-6 um) are deparaffinized, stained, counterstained, dehydrated, cleared and mounted. Block the sections with 10% normal rabbit serum to inhibit non-specific binding. Dilute the Protein G purified sheep anti-ET-Br or anti-ET-Ar to 10 ug IgG/ml in PBS containing 1.0% normal rabbit serum.Perform the immunoreactivity by using a peroxidase-coupled rabbit anti-sheep IgG.


IMMUNOPRECIPITATION OF ET-A and ET-B receptors

Prepare cell lysates (-5mg) by homogenizing tissues in 2mL RIPA (50mM Tris, pH = 7.5, 150mMNAC1, 1% NP-40, 0.5% deoxycholic acid,0.1% SDS, 2 mM EDTA, 10 mM NaF, 10 ug/ml leupeptin, 10 ug/ml pepstatin, 20 ug/ml aprotinin). Centrifuge the homogenate for 10 minutes at 1000xg. Dilute 500ul of the cell lysate 1:1 in RIPA buffer. Immunoprecipitate the receptors with 4ug of appropriate antibody for 1 hour at room temperature. Collect the precipitin with Protein G Plus Agarose and wash 3 times with 1.0 ml RIPAbuffer. Suspend the final pellet in 40 ul 2X SDS loading buffer,heated at 100'C for 5 minutes and recentifuge for 5 minutes in a microcentrifuge. Load 40uls of the supernatent onto a 8-16% gradient SDA-PAGE minigel. After electrophoresis, transfer the proteins to a nitrocellulose filter and block overnight in blocking buffer (3% BSA, 10 mM Tris pH 7.5, 100 mM NaC1, & 0.1% Tween-20). React the transferred proteins with Endothelin A & B receptor antibodies diluted 1:400 in blocking buffer for 1 hour at 25'C. Then add rabbit anti-sheep IgG HRP (KPL) diluted 1:5000 in blocking buffer for 1 hour at 25'C. Wash the nitrocellulose filter extensively. Detect the Endothelin A & B proteins using the Enhanced Chemiluminescent detection system by Amersham (tm).

Saponin Method:

Dulbecco's PBS (without Mg2+ or CA2+)

1% heat inactivated FCS (or RIA grade BSA)

0.1% w/v sodium azide

0.1% w/v saponin (Sigma cat#S-7900)

Adjust pH buffer to 7.4-7.6 and filter

see ref:

1)Sander, B., J. Andersson and U. Andersson, 1991:Assessment of cytokines by immunofluorsecence and the paraformaldehyde-saponin procedure:Immunol Rev 119:65-93

2) Anderson, U. and J. Andersson 1994. Immunolabelling of cytokine producing cells in tissues and suspension. In Cytokine Producing Cells eds. D. Fradelizie and D. Emelie. INSERM, Paris.32-49.

-immunogen sequence: AC-s-s-h-v-e-d-f-t-p-f-p-g-t-e-f-c-amide

-antibody purified by Protein G aff chromatography

-use: western blot, Eia

-immunogen: endothelin B-n-terminal receptor-GST fusion protein

                      sequence: G S N S S L M R F R T A E V

-antibody purified by Protein G affinity chromatography

-suitable for EIA, western blot  (also humna/mouse/rat reactive)

-immunogen: Endothelin B receptor peptide-KLH conjugate

                      sequence: Ac-g-s-n-s-s-l-m-r-f-r-t-a-e-v-c-amide

-antibody purified by Protein G chromatography

-use: western blot, Eia

RDI Divison of researchd Industries Intl

San Jose, 95123 CA Snell ave 658

USA

or (800) 370-2222

EMAIL:margaret@cellular-research.com

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