rev:    October 11, 2004

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Cytokines, Growth Factors & Chemokines:ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of Fitzgerald Industries Intl  offers a wide line of recombinant growth factors, cytokines and chemokines and 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.


Chemokine Receptor polyclonals

-see also anti-Goat secondary antibodies

see sample WB and histo protocols below


$565.00/vial    email for specs not linked (in process)

blocking peptides $250.00/100ug
Specificity Host Size Catalog#
Human CCR-1 rabbit 100ul RDI-CCR1abrX
Mouse CCR4 rabbit 100ul RDI-MCCR4abrX
Human CCR-5 Goat 200ug RDI-CCR5NabGX
Human CXCR-4 Goat 200ug RDI-CXCR4abGX
Mouse CXCR-4 Goat 200ug RDI-MCXCR4abGX
Human CCR4 Goat 200ug RDI-CCR4abGX
Human CCR2 Goat 200ug RDI-CCR2abGX
Human CCR3 Goat 200ug RDI-CCR3abgX
Human CCR8 n term Goat 200ug RDI-CCR8NabGX
Human CCR8 2nd

extracellular domain

Goat 200ug RDI-CCR8XabgX
Mouse CCR4 N term Goat 200ug RDI-MCCR4abgX
Mouse CCR8 extracellular domain Goat 200ug RDI-MCCR8XabgX
Human CCR9 extracellular domain Goat 200ug RDI-CCR9XabgX
Mouse CCR9 extracellular domain Goat 200ug RDI-MCCR9XabgX
Human CCR-10 extracellular domain Goat 200ug RDI-CCR10XabgX
Mouse CCR-10 extracellullar domain Goat 200ug RDI-MCCR10XabGX
Human CCR-11 ext Goat 200ug RDI-CCR11XabGX
Human CCR-6 extracellular domain Goat 200ug RDI-CCR6XabGX
Human CCR-D6 Goat 200ug RDI-CCRD6abgX
Mouse CCR-7 extracellular domain Goat 100ug RDI-MCCR7XabgX
Mouse CCR-D6 Goat 200ug RDI-MCCRD6abGX
Human Toll Receptor 2 ext domain Goat 200ug RDI-TOLLR2XabgX
Human Toll Receptor 5 ext domain Goat 200ug RDI-TOLL5RXabgX


Rabbit anti-Human CCR-1  F(ab')2

cat# RDI-CCR1abRX   $562.00

Presentation: 100ul of peptide affinity purified F(ab')2 IgG in   PBS with 1 mg/ml BSA and 0.1% sodium azide.

host: rabbit IgG

Antigen: Synthetic Peptide

Sequence of Peptide NH2-TEDYDTTTEFDYGDATPC

Specificity: Peptide sequence shares less than 50% amino acid identity to other Chemokine receptors in this region. Since the peptide sequence shares less than 50% amino acid identity to other chemokine receptors in this region, this antibody only recognizes human receptor.

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Application: (Working Dilutions)

    Western Blotting and Immunohistochemistry on Acetone fixed cells.

    ELISA/peptide : 1:50,000

    Western Blot: 1:1000

     Immunocytochemistry: 1: 300

      Immunohistochemistry: 1: 300

Quality Control Test for Each Lot: Western Blot on membranes from human peripheral blood leukocytes or human T-cell line Jurkatt.

References: Neote K, DiGregorio D, Mark J. Y.,Horuk. R.and Schall T.J. 1993. "Molecular Cloning, Functional Expression, and Signaling Characteristics of a C-C Chemokine Receptor." Cell. 72, 415-425.

Su S.B., Mukaida. N., Nomura. H and Matsushima. K. 1996. "Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1expression on various types of leukocytes."

J Leukoc. Biol 60(5), 658-666.

For research Use Only


Goat anti-human CCR2 (affinity purified) Antiserum (affinity purified) to C-C Chemokine Receptor 2 (human)

Cat# RDI-CCR2abGX  $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide CGEEVTTFFDYDYGAPCHKFDVKQ corresponding to the N-terminal extracellular domain of human CCR2.

Specificity: Peptide sequence is < 50 % identical to other chemokine receptors in this region. Cross reactivity to mouse CCR2 not tested.

Applications: ELISA 1:60000

                      Immunoblotting >1:1000

                     Immunocytochemistry >1:400

                     Immunohistochemistry >1:200

                    pos control spleen cells

                   Flowcytometry N. D

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature References: [1] Charo,I.F., Myers,S.J., Herman,A., Franci,C., Connolly,A.J. and Coughlin,S.R. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternate splicing of the carboxyl-terminal tails. Proc. Natl. Acad. Sci. U.S.A. 91, 2752-2756 (1994)

For research Use Only-not for use in or on humans or animals


Goat anti-human CCR3 affinity purified

cat# RDI-CCR3abGX    $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Immunogen: 28 amino acid synthetic peptide   CVETFGTTSYYDDVGLLCEKADTRALMA

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. CCR3 is the receptor for eotaxin and a novel CC Chemokine, leukotactin-1. CCR3 is expressed on eosinophils, TH2 cell subsets, basophils, and mast cells, and plays a key role in allergic diseases.

Applications: ELISA to peptide 1:60000

                       Immunoblotting 1:1000

                       Immunocytochemistry 1:300

                         Immunohistochemistry 1:300

Positve Control: human peripheral blood cells.

Storage/Stability: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature  References: -Auger, G.A., et al. 2002. Eur. J. Immunol. 32, 1052;

                                      -Humbles, A.A., et al. 2002. Proc. Natl. Acad.Sci. USA 99, 1479

                                      -Power, C.A., et al. 1995. J. Biol. Chem. 270, 19495

Background: Human CCR3 is a receptor for CC chemokine eotaxin-1 and oetaxin-2. CCR3 is constitutively expressed on eosinophils, small intestine and colon. The human CCR3 protein contains 356 amino acid. Antibody to human CCR3 receptor was generated to a synthetic peptide derived from the amino terminal extracellular domain of the human CCR3 receptor. Goat anti-CCR3 IgG antibody binds to human eosinophils at a dilution of > 1.500.

For Research Use Only


Goat anti-human CCR4 (affinity purified)   Antiserum (affinity purified) to C-C Chemokine Receptor 4 (human)

Cat# RDI-CCR4abgX   $562.00

Presentation: 200ug of peptide affinity purified IgG in PBS at 1.0mg/ml with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat

Immunogen: 28 amino acid synthetic peptide  L D E S I Y S N Y Y L Y E S I P K P C T K E G I K A F G E.

derived from the amino terminal extracellular domain of the human CCR4 receptor.

Specificity: Peptide sequence is <50% identical to other Chemokine receptors in this region.

Applications:

ELISA >1: 100000

Western Blot >1: 1000

Immunocytochemistry (acetone fixed cells) >1: 400

Immunohistochemistry (paraffin sections) >1: 200

Not tested for Flowcytometry

Introduction: Human CCR4 is a receptor for macrophage inflammatory protein 1 alpha and Rantes and is expressed on human basophils and 20% of the human adult peripheral blood CD4+ cells. The human CCR4 protein contains 360 amino acids that is 85% identical to mouse CCR4 receptor and contains three glycosylation sites. human CCR4 transcripts are expressed in the thymus and peripheral blood leukocytes.

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature References: Power., CA etal. 1995 Molecular Cloning and Functional Expression of a Novel CC Chemokine

Receptor cDNA from a Human Basophilic Cell Line. J Biol. Chem. vol. 270 :33: 19495-19500.

For research Use Only


Goat anti-mouse CCR4 N-Terminal Antiserum (affinity purified) to C-C Chemokine Receptor   4 (mouse).

cat# RDI-MCCR4abGX    $562.00

Presentation: 200ug peptide affinity purified GOAT IgG in PBS with 1 mg/ml BSA and buffers containing 0.1% sodium azide.

Host: Goat

Antigen: Synthetic peptide DTTQDETVYNSYYFYESMPC corresponding to aa 8-26 of mouse CCR4. This seequence corresponds to amino acid residues 8 - 26 of mouse CC chemokine receptor CCR4.

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. The antiserum recognizes mouse CCR4. No cross-reactivity to human CCR4. CCR4 is selectively expressed on Th2-type T cells and as been shown to be responsible for Th2-dominant immune responses. Its ligand is the thymus and activation-regulated chemokine TARC No cross-reactivity to human CCR4.

Applications: ELISA >1:50000

                       Immunoblotting >1:1000

                       Immunobytochemistry >1:500 (mouse spleen)

                       Flow Cytometry 1-3ug/10^6 cells/50-100ul

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature

References: Molecular cloning of mouse CC CKR-4 and high affinity binding of chemokines to murine and human CC CKR-4: A.J. Hoogewerf, D.Black, A.F. Proudfoot, T.N.C. Wells, and C.A. Power; BBRC 218, 337 (1996)

For Research Use Only


Goat anti-human CCR5 (affinity purified)   Antiserum (affinity purified) to C-C Chemokine   Receptor 5 (human)

cat# RDI-CCR5NabgX    $562.00

Presentation: 200ug of peptide affinity purified IgG in PBS with 1 mg/ml BSA and 0.1% sodium azide.

Antigen: Synthetic peptide (NH2-YQVSSPIYDINYYTSEPCQKINVKQIAA) corresponding to the N-terminal sequence of human CCR5.

Specificity: Binds to CCR 5 receptor on human peripheral blood leukocytes as determined by immunocytochemistry.

Applications:

  ELISA 1:50000

  Western Blot 1:1000

   Immunocytochemistry: 1: 300 (pos control human peripheral blood)

    Immunohistochemistry 1: 300 (human spleen)

Background: The HIV receptor is composed of two molecules, the CD4 binding receptor and a co-receptor. The chemokine receptor CCR5 is one of the co-receptors used by primary isolates of HIV-1. Extracellular domains of CCR5 are important for HIV entry into the target cells.

Storage/Stability: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References:

1. A RANTES, MIP-1a, MIP-1b receptor as fusion cofactor for macrophage-tropic HIV-1: Alkhatib et al.; Science 272, 1955 (1996)

2. Muliple Extracellular Domains of CCR-5 contribute to human immunodeficiency virus type 1 entry and fusion: J.Virol. 71, 5003 (1997)


Goat anti-mouse CCR4  N-Terminal   Antiserum (affinity purified) to C-C Chemokine Receptor   4 (mouse).

cat# RDI-MCCR4abGX   $562.00

Presentation: 200ug peptide affinity purified GOAT IgG in PBS with 1 mg/ml BSA and buffers containing 0.1% sodium azide.

Host: Goat

Antigen: Synthetic peptide DTTQDETVYNSYYFYESMPC corresponding to aa 8-26 of mouse CCR4. This seequence corresponds to amino acid residues 8 - 26 of mouse CC chemokine receptor CCR4.

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. The antiserum recognizes mouse CCR4. No cross-reactivity to human CCR4. CCR4 is selectively expressed on Th2-type T cells and has been shown to be responsible for Th2-dominant immune responses. Its ligand is the thymus and activation-regulated chemokine TARC No cross-reactivity to human CCR4.

Applications: ELISA >1:50000

                      Immunoblotting >1:1000

                      Immunobytochemistry >1:500 (mouse spleen)

                      Flow Cytometry 1-3ug/10^6 cells/50-100ul

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature References: Molecular cloning of mouse CC CKR-4 and high affinity binding of chemokines to murine and human CC CKR-4: A.J. Hoogewerf, D.Black, A.F. Proudfoot, T.N.C. Wells, and C.A. Power; BBRC 218, 337 (1996)

For Research Use Only


Goat anti- Human CxCR4 (affinity purified)   N-terminal - extracellular domain

Cat# RDI-CXCR4abGX  $562.00

Presentation: 200ug of peptide affinity purified IgG in PBS at 1.0mg/ml with 1 mg/ml BSA and 0.1% sodium azide. Antibody in formulation without azide for neutralization studies is also available.

Host: Goat

Antigen: TEEMGSGDYDSMKEPCFREENANFNKC corresponding to N-terminal extracellular domain of human CxCR4 receptor.

Specificity: Binds to CXCR 4 receptor on human spleen leukocytes as determined by immunocytochemistry.

Applications: ELISA: 1:50,000

                      Western Blotting titer 1:1000

                       Immunocytochemistry 1:300 (human spleen)

                        PARAFFIN SECTIONS

Storage: Freeze at -80 °C. Stable for at least 3 years when   stored at -80 °C. Avoid freeze/thaw cycles.

References:

1. Bleul, C.C etal 1996. The lymphocyte chemoattractant, SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382 : 829-833.

2. Loetscher M. etal. 1994. Cloning of a human seven transmembrane domain receptor, LESTR, that is highly expressed in leukocytes. J Biol. Chem. 269, N 1: 232-7

For research Use Only


Goat anti- mouse to CxCR4 (affinity purified)   N-terminal - extracellular domain

Cat# RDI-MCXCR4abGX

Presentation: 200ug of peptide affinity purified IgG in PBS at 1.0mg/ml with 1 mg/ml BSA and 0.1% sodium azide. Antibody in formulation without azide for neutralization studies is also available.

Host: Goat

Antigen: NH2-YSEEVGSGDYDSNKEPCFRDENVHFNR

Specificity: Binds to CXCR 4 receptor on mouse spleen leukocytes as determined by immunocytochemistry.

Applications:

     ELISA: 1:50,000

     Western Blotting titer 1:1000

      Immunocytochemistry 1:300 (mouse spleen)

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References: 1. Bieniasz et.al. 1997. Murine CxCR-4 is a functional coreceptor for T-cell tropic and dual tropic strains of human immunodeficiency virus type 1. J Virol. 71: 7097.

2. Heesen, M. etal. 1996. Cloning of the mouse fusin gene. homologue to a human HIV-1 cofactor. J Immunol. 157: 5455.

For research Use Only   see also other anti-cxcr4 ab (fusin)


Goat anti-Human CCR-6 Extracellular Domain   Human

cat# RDI-CCR6XabGX   $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide YFVSVNTSYYSVDSEMLLCSLQEVRQFSR corresponding to a.a. 18-46 of human beta chemokine receptor D6.

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. CCR6 is a G-protein-coupled receptor that binds MIP-3a. It is expressed on T cells, B cells, and subsets of dendritic cells, and has been implicated in the adaptive immune response. The antiserum recognizes human CCR6.

Applications: ELISA                           1: 75000

                      Western blot                  not tested

                       Immunocytochemistry   1:300

                       Immunohistochemistry 1:250  (pos control paraffin section human spleen)

                       Flowcytometry 1-3ul/million cells

Quality Control: Immunocytochemistry on human spleen or peripheral blood.

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References: Baba, M. etal, Identification of CCR6, the specific receptor for a Novel Lymphocyte directed CC Chemokine LARC. J. Biol. Chem. 272 (23), 14893-14898 (1997)

For Research Use Only


Goat anti-Mouse C-C Chemok. Receptor-7 Ext.Cell.Dom

cat# RDI-MCCR7XabgX   $562.00/100ug

Presentation: 100ug of peptide affinity purified IgG antibody in PBS with 1 mg/ml BSA and 0.1 % sodium azide.

Antigen: Synthetic peptide corresponding to aa 2-22 of mouse CCR7.

Specificity: Peptide sequence is < 50 % identical to other mouse chemokine receptors in this region.

Applications:

  ELISA 1:100000

  Western Blot 1:1000

  Immunocytochemistry 1:500 (mouse spleen)

  Immunohistochemistry 1:250

  Flowcytometry not tested

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature  References: Cloning of human and mouse EBI1, a lymphoid-specific G-protein-coupled receptor encoded on human chromosome 17q12-q21.2. Genomics 23 (3), 643-650 (1994)

For  In Vitro Research Use Only


Goat anti-Mouse C-C Chemok. Receptor-8 Ext.Cell.Dom

cat# RDI-MCCR8XabgX    $562.00/100ug

Presentation: 200ug of peptide affinity purified IgG antibody in 0.1ml (2mg/ml) PBS (1mM KHPO4, 140mM NaCl pH7.2)   with 1mg/ml BSA and 0.1 % sodium azide.

Antigen: Synthetic peptide PNVTMTDYYPDFFTAPCDAEFLLRGSM   corresponding to a 7-33 of mouse CCR8

Specificity: Peptide sequence is < 50 % identical to other mouse chemokine receptors in this region. The antiserum recognizes mouse CCR8. Not tested for cross-reactivity to human CCR8.

Applications:

      ELISA 1:100000

      Western Blot 1:1000

       Immunocytochemistry 1:400 (mouse spleen)

        Immunohistochemistry 1:400

        Flowcytometry not tested

Positive Control: mouse spleen (paraffin sections)

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature

References: 1. Zingoni, A. et. al. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells J. Immunol. 161 (2), 547-551 (1998)

For Research Use Only


Goat anti-human C-C chemokine Receptor-9, Ext.Cell.Dom

cat# RDI-CCR9XabgX    $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide IPNMADDYGSESTSSMEDYVNFNFTDFYC corresponding to aa 10-37 of human CCR9.

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. The antiserum recognizes human CCR9 and is likely to cross react to mouse CCR9 with approximately 50% less titer .

Applications:

ELISA (human CCR peptide)1: 75000

Western Blotting 1:1000

Immunocytochemistry 1:300

Immunohistochemistry 1:250

Flowcytometry-not tested

Quality  Control: Immunocytochemistry on human thymus, spleen or   MOLT4 cell line

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References: [1] Zaballos,A.,. et. al. identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. J. Immunol. 162 (10), 5671-5675 (1999)

[2] Yu, et. al. "CCR9A and CCR9B: Two Receptors for the Chemokine CCL25/TECK/Ckbeta-15 That Differ in Their Sensitivities to Ligand. J Immunol. 164, 1293-1305, 2000

For Research Use Only


Goat anti-mouse CCR9

cat# RDI-MCCR9XabGX   $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide IPGMFDDFSYDSTASTDDYMNLNFSSFF corresponding to aa 10-37 of mouse CCR9.

Specificity: Peptide sequence is < 50 % identical to other mouse chemokine receptors in this region. The antiserum recognizes mouse CCR9 and is likely to cross-react to human CCR9 with approximately 50% less titer.

Applications:

ELISA 1: 100000

Western blot 1:1000

Immunocytochemistry 1:500

Immonuhistochemistry 1:250

Flowcytometry 1:200

Quality Control: Immunocytochemistry on mouse thymus or lymphnodes.

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References: Zaballos,A.,. et. al. identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. J. Immunol. 162 (10), 5671-5675 (1999)

For Research Use Only


Goat anti-mouse CCR10

cat# RDI-MCCR10XabGX    $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide KPTEQVSWGLYSGYDEEAYSVGPLPELC corresponding to aa 4-31 of mouse CCR10.

Specificity: Peptide sequence is < 50 % identical to other mouse chemokine receptors in this region. CCR10 is the receptor for the skin-associated CC chemokine CCL27 (CTACK) as well as a novel chemokine CCL28 that displays chemotactic activity for resting CD4+ and CD8+ T cells. The antiserum recognizes mouse CCR10 and has minimal cross-reactivity to human CCR10.

Applications: ELISA 1: 100000  

                       Western blot 1:1000

                        Immunocytochemistry 1:500

                        Immonohistochemistry 1:250

                        Flowcytometry not tested

Quality  Control: Immunocytochemistry on mouse lymphnodes or small intestine

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

References: 1] Zaballos,A., et.al. Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. J. Immunol. 162 (10), 5671-5675 (1999).

[2] Wang, W., et al. 2000. J. Biol. Chem. 275, 22313.

For Research Use Only


Goat anti-Human CCR10

cat# RDI-CCR10XabGX  $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide VSWGHYSGDEEDAYSAEPLPELC corresponding to aa 9-31 of human CCR10.

Specificity: Peptide sequence is < 50 % identical to other mouse chemokine receptors in this region. CCR10 is the receptor for the skin-associated CC chemokine CCL27 (CTACK) as well as a novel chemokine CCL28 that displays chemotactic activity for resting CD4+ and CD8+ T cells. The antiserum recognizes human CCR10 (not tested with mouse CCR10)

Applications: ELISA 1: 75K

                     Western blot 1:1000

                     Immunocytochemistry 1:250

                      Immunohistochemistry 1:250

                     Flowcytometry not tested

Quality Control: Immunocytochemistry on paraffin section of human spleen

Storage: Freeze at -70 °C. Stable for at least 3 years when stored at -70 °C. Avoid freeze/thaw cycles.

References: 1] Zaballos,A., et.al. Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. J. Immunol. 162 (10), 5671-5675 (1999).

[2] Wang, W., et al. 2000. J. Biol. Chem. 275, 22313.

For Research Use Only


Goat anti-human C-C chemokine Receptor-11,   Ext.Cell.Dom (Human/Mouse)

Cat #: RDI-CCR11XabGX   $562.00

Presentation: 200ug of peptide affinity purified IgG in 0.1ml (2mg/ml) 10mM KHPO4, 140mM NaCl, pH7.2 with 1 mg/ml BSA and 0.1% sodium azide.

Host: Goat IgG

Antigen: Synthetic peptide EQNQSTDYYYEENEMNGTYDYSQYELIC corresponding to aa 4-31 of human CCR11.

Specificity: Peptide sequence is < 50 % identical to other human chemokine receptors in this region. Will cross-react to mouse CCR11.

Applications: ELISA 1:75000

                       Western Blot 1:1000

                        Immunocytochemistry 1:500

                        Immunohistochemistry 1:250 (human lung tissue)

                        Flowcytometry N. D.

Storage: Freeze at -80 °C. Stable for at least 3 years when stored at -80 °C. Avoid freeze/thaw cycles.

Literature

References: 1] Schweickart,V.L., etal. CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines. J. Biol. Chem. 275 (13), 9550-9556 (2000)

For Research Use Only-not for use in or on humans or animals

Immunohistochemistry with antibody to Human CCR11 on paraffin section of mouse Lung


SAMPLE PROTOCOLS:

Immunohistochemistry on Paraffin sections:

Paraffin sections of tissues fixed in 10% formalin were used for QC of the immunodetect antibodies. De-wax the sections and block the endogenous peroxide by incubating the sections with 3% H2O2 for 15 minutes at room temperature (RT). Wash the sections with PBS and incubate with 2% normal rabbit serum at room temperature to block the non-specific binding sites. Wash the section with PBS and incubate with primary antibody (anti-chemokine receptor) at recommended dilution for 60 minutes at RT. Wash the slides and incubate at RT with biotinylated rabbit anti-goat IgG H+L (Vector Labs) at a 1:200 dilution for 30 minutes. Wash the slides and incubate with HRP-Streptavidin (Vector Labs) 1:400 dilution for 30 minutes. Wash the slides and incubate with DAB (Sigma) for 5-7 minutes at RT. Wash slides with PBS and counter stain with hematoxylin (DAKO).

Immunocytochemistry & Immunohistochemistry

Immunocytochemistry on cytospun cells or frozen tissues section:

Fix the cells cytospun on slides or frozen tissue section in cold acetone (acetone prechilled to -20C) for 2-5 minutes. Let the slides dry and then block endogenous peroxide activity by treating the slides with peroxide blocker (3% hydrogen peroxide) for 10 minutes. If the secondary antibody is from rabbit then cells should be incubated with 2% normal rabbit serum in PBS. Wash the slide in PBS x 5 then incubate with the antibody at appropriate dilution in PBS containing Ig free 2% BSA or 2% milk for 30-45 minutes at room temperature, wash 5 times with PBS and then incubate with second HRP-conjugated antibody at a dilution optimized previously for 30 minutes at room temperature (If using primary cells from lymphoid organs containing B cells as well the secondary antibody must be specifies specific to minimize staining of B cells by secondary antibody alone). Wash 5 times with PBS and develop the stain by adding the DAB or AEC and counter stain with hematoxylene.

Western Blotting

SDS-PAGE: A NOVEX gel system or any other gel system based on Laemmli's SDS-PAGE system is recommended. If protein of interest is expressed at very low levels in the cells and/or only a very small percentage of cells in the tissue express the desired protein, then a higher amount of total tissue lysate or crude membrane lysate in the range of 100-150 ug of protein per lane is recommended. SDS-PAGE gels used should contain appropriate percent acrylamide to provide the best separation of proteins in the molecular weight range of the desired protein to be detected. We strongly recommend separating proteins on SDS-PAGE gels at 15-20 mA constant current when 100-150 ug of protein is loaded in each lane to ensure clear separation. SDS-PAGE can be run overnight at 5-7 mA constant current and next day in the morning the current should be increased to 20 mA until the dye front reaches the bottom of the gel. This will tighten the bands in the gel.

Transfer of Proteins to Nitrocellulose: When transferring hydrophobic integral cell membrane proteins Tris-Glycine buffer containing 1 x SDS running buffer with 10 to 20% methanol should be used to facilitate transfer of proteins like CCRs that have 7 hydrophobic domains. Tris-Glycine buffer with 10-20% methanol is adequate to transfer most secreted proteins. Transfer the proteins following the instructions provided by the manufacturer of the transfer apparatus. For the semi-dry transfer system we used, protein transfer was done at 150 mA constant current for 60-90 minutes.

Immunodetection: After the transfer, air dry the Nitrocellulose membrane on a paper towel. Rinse the membrane in Tris buffered saline with 0.1% Tween 20 (20 mM Tris, 150 mM NaCl pH 7.5-8.0) for 5 minutes. Block the membrane in 5% milk in TBS-Tween for a minimum of 1 hour. Incubate with anti-chemokine receptor antibody diluted in 1-2% milk in TBS-Tween at room temperature for 2 hours or overnight at 4 C. Wash the membrane x 5, 5 minutes each with TBS-Tween containing 300 mM NaCl (Note TBS-Tween indicated above contains 150mM NaCl. Higher salt is used to facilitate removal of non-specifically bound antibody). Incubate with HRP conjugated secondary antibody at appropriate dilution in 1-2% milk in TBS-Tween containing 150 mM NaCl for a minimum of 1 hour. If using B cells the secondary antibody should be species specific to minimize detection of Ig expressed by B cells by secondary antibody. Wash the membrane x 5, 5 minutes each with TBS-Tween containing 300 mM NaCl. Visualize the bands using the ECL system of Amersham-Pharmacia.


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