rev:   March 1, 2005

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CD CLUSTERED ANTIBODIES  

(anti-Human and others as indicated)

NOTE: Please inquire for bulk or custom formulations (without preservative or carrier).

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (flow cytometry, neutralization, blotting, histochemistry, 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. All products are for in vitro research use only-not for use in or on humans or animals-not for use in diagnostics. Price/availability/specifications subject to change without notice.

CD51  Antibodies

Mouse anti-human CD51  antibodies

-3 clones 

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-CD51abm-W          P2W7                            V             mIgG1      purified     $312.00

RDI-CD51abm-WBT     P2W7                            V             mIgG1       Biotin       $375.00

RDI-CD51abm-WFT    P2W7                            V             mIgG1       FITC          $375.00

RDI-CD51abm-WPE    P2W7                            V             mIgG1      PE               $469.00

RDI-M1541clb               CLB-M9                        IV           mIgG1      purified     $375.00

RDI-CBL490                  13C2                             IV           mIgG1      purified      $375.00

RDI-CBL490FT               " "                                 "               "             FITC        $375.00

RDI-CBL490PE              " "                                  "               "             PE            $469.00

RDI-CD51abrX          rabbit poly                

cat# RDI-CD51abm-WPE   $469.00/vial

Presentation: 120 tests in 0.2ml (50 mM Sodium Phosphate pH 7.5, 500 mM Potassium Chloride, 150mM NaCl, 15% Glycerol, 0.2% BSA, 0.04% NaN3 (as a preservative).

Clone: P2W7

Isotype: Mouse IgG1k

Immunogen: Human ocular melanoma cell line (V+B2)

PRODUCTION: Protein A purified antibody from tissue culture supernatant was conjugated to R-Phycoerythrin through a sulfo-ester linkage. Unconjugated antibody was removed using size exclusion chromatography. Antibody conjugate is at 0.5 mg/ml with an A565/A280 ratio of 2.77.

DILUTION: 1:50 (80ul/test)

STORAGE: Store at 2 - 5 DEg C. Do not freeze! Protect from light.

Sample Protocol: 500K cultured UM-SCC (squamous cell carcinoma) cells were harvested by trypsinization, washed and preincubated 5 minutes with 20 ul of 250ug/ml human IgG (To block nonspecific binding), after which they were incubated 45 minutes on ice with 80 ul of anti-CD51/R-PE at a 1:50 dilution. Cells were washed three times, fixed and analyzed using a BD FACstar plus. Cells stained positive with a mean shift of 1.86 log10 fluorescent units when compared to a Mouse IgG1/R-PE negative control (cat#MIGG1PEX2 $188.00) at a similar concentration. Binding was 90% blocked when pre incubated 10 minutes with 20ul of 0.5 mg/ml anti-CD51 antibody (unconjugated)

Background: Human CD51 is an integrin alpha v chain which can associate with five different integrin beta chains to form various adhesion molecules. The alpha vbeta 3 integrin complex binds vitronectin at the RGD sequence and also can bind to fibrinogen, von Willebrand factor, thrombospondin, fibronectin, osteopontin and collagen. Antibody P2W7 recognizes the alpha v molecule of 160 kd.

References: R.O. Hynes (1992) Cell 69: 11-25. Leukocyte Typing V (S.F. Schlossman, et al, eds.) Oxford University Press, Oxford (1995) p. 1663-1664.

For Research Use Only-Not for use in diagnostic procedures

R-Phycoerythrin (R-PE) is covered under patents: U.S. 4,520,110; European 76,695 and Canadian 1,179,942.

Also available: (Bulk quotes on request)

non conjugated cat#RDI-CD51abm-W             $312.00/100ug

FITC conjugated: cat#RDI-CD51abm-WFT    $375.00/120T

Biotin conjugated: cat#RDI-CD51abm-WBT  $375.00/100ug

PE conjugated: cat#RDI-CD51abm-WPE       $469.00/100T

PRODUCT CODE :RDI-CBL490     $375.00/vial 200ug

also available FITC labeled, cat#RDI-CBL490FT    $375.00/100T

                         PE labeled, cat#RDI-CBL490PE    $469.00/100T

CLONE                                     13C2

ISOTYPE                                   IgG1

IMMUNIZING ANTIGEN          Human osteoclastoma tissue

SOURCE                                     Tissue culture supernatant

PURIFICATION METHOD         Protein A affinity chromatography

SPECIFICITY This antibody recognizes a 110/90 kDa antigen known as the a chain of the vitronectin receptor (VNR). This forms a complex with the CD61 antigen and mediates cell adhesion to RGD (arg-gly-asp) containing sequences in vitronectin, von Willebrand factor, fibrinogen and thrombospondin. This antibody reacts with osteoclasts, placenta and melanoma cell lines, some endothelial cells, smooth muscle cells and fibroblasts, and weakly with platelets.

APPLICATIONS * Bone resorption modulation* Osteoclast identification* Purification of the vitronectin receptor* Malignant melanoma identification (and possible treatment)* The antibody is only reactive on frozen sections

REFERENCES9712CBL 490 CONT'D Cancer Research 45, 5663-69 (1985)J. Cell Biol. 109, 1817 (1989)Leucocyte Typing IV, Oxford University Press (1989)

LOT SPECIFIC DATA

PRESENTATION The monoclonal is presented at a concentration of 200µg/2ml in phosphate buffered saline containing 10mM sodium azide and 1mg/ml bovine serum albumin. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE For use within 1 month of purchase store at +4oC, for long term storage aliquot antibody into small volumes and store at -20oC.

For Research Use Only. Not supplied for use in diagnostic or therapeutic procedures.

Also available in milligram sizes with or without BSA or azide (approx $1000.00/mg)

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD51- antigen (VNR-alpha-chain), which can form distinct complexes with:

- the CD61-antigen (gpIIIa or 3-chain) resulting in the alpha- 3 complex, which is expressed on human platelets;

- the 5 integrin, resulting in the alpha- 5 complex, which is expressed on human monocytes and macrophages;

- the 1 integrin, resulting in the alpha- 1 complex.

Molecular mass 25, 125 kDa.

Application Functional studies on cells.

Methods Indirect immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

References 1. Vries, J.E. de et al., Int. J. Cancer, 38, 465 (1986).

APPLICATION  FOR AN INDIRECT IMMUNOFLUORESCENCE TECHNIQUE

Reagents

- FITC-conjugated goat anti-mouse immunoglobulin antiserum (M1204).

  Remove aggregates by centrifugation at 1000 x g for 10 minutes.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumin (BSA).

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2% BSA.

- Microtiter plates (96 wells, V bottom) or tubes.

Procedure

1    Fixate the cells with PFA 1% and wash them with buffer.

2    Prepare a mononuclear cell suspension with a concentration of 1 x 10 7 cells/ml in buffer.

3    Add 45 µl of cell suspension to the microtiter wells or tubes.

4    Add 5 µl monoclonal antibody to the microtiter wells or tubes and mix gently.

5    Incubate for 30 minutes at 2-8 C.

6   Add 100 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

7   Aspirate the supernatant from the cell pellet and resuspend the cells.

8   Add 200 µl buffer to the microtiter wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

9   Aspirate the supernatant from the cell pellet and resuspend the cells.

10  Add 50 µl FITC-conjugated goat anti-mouse immunoglobulin serum, solid phase adsorbed with human                                 immunoglobulins, diluted 1:80.

11  Incubate for 30 minutes at 2-8 C.

12  Repeat step 6-9.

13  Add 200 µl buffer.

14  The number of positive cells can be determined by flowcytometry analysis or by means of fluorescence microscopy.

NOTE: Care should be taken when drawing blood to avoid activation of platelets.

Platelets activation markers are weakly expressed on platelets prepared from directly fixed normal whole blood.

The expression is maximal on thrombin (1 U/ml) stimulated washed platelets.

FOR IN VITRO RESEARCH USE ONLY

Catalog#: RDI-CD51abrX     $438.00

Package Size: 100ul neat sera with 0.05% NaN3

Species: rabbit

lot#

Immunogen: Synthetic peptide (KRVRPPQEEQERE) derived from the COOH terminal sequence (cytoplasmic domain) of the human integrin alphaV subunit acids 1022-1034).

Reactivity: Integrin alphaV. Specificity determined by immunoprecipitation from a detergent extract of 35S methonine labeled endothelial cells. No cross-reactivity to alpha1, alpha2, alpha3, alpha4, or alpha6 integrin subunits was detected. The mature alphaV protein (150kDa) is composed of a disulfide bonded heavy (125 kDa) and a light (25 kDa) chain derived from a proteolytically cleaved precursor (Suzuki, 1987). The epitope recognized by the antibody is located on the COOH terminal portion of the light chain. By western blot under reducing conditions the antibody detects two major bands at approximately 25 and 27 kDa corresponding to the light chain. The slight differences in sizes of the light chains is thought to be caused by proteolytic cleavage at two alternative sites on the precursor. Similar sites have been reported for alpha6, alpha3, and alphalIbeta (Hogervorst, 1991).

Species : Reacts with horse, Goat, Human,Mouse, Pig, Sheep, Rat, Hamster and Chicken. Reactivity with other species has not been confirmed.

Uses: -Western blot: 1:5000. The antibody recognizes a single band at approximately 150 kDa when denatured, but not reduced (Hirsch, 1994). Under reducing conditions this antibody recognizes two major bands of 25 and 27 kDa and, to a lesser extent, the full length 150 kDa band.

-Elisa: 1:1000

Immunoprecipitation: 5 uL of antibody is sufficient to precipitate alphaV/beta1 from 5x106 cells.

Immunohistochemistry: 1:1000 (Suggested for use an acetone fixed tissue only.)

Titers must be optimized for each application

general ref: -Hirsch, et al. (1994). alphaV integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development. Develop. Dynamics 201:108-120.

-Hogervorst, F. et al. (1991). Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes. Eur. J. Biochem. 199:425-493.

Storage: Store at -20 DEG C in undiluted aliquots. For extensive dilutions, add protein containing or stabilizing medium. Avoid frequent freeze thaw cycles.

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

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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