rev:   April 23, 2002

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Anti-MOUSE
CD CLUSTERED ANTIBODIES  

RDI Divison 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.


Anti-MOUSE  CD140a Antibodies


DATA SHEET: Rat anti-Murine PDGFR Alpha Chain (CD140a)

Catalog#:    RDI-MCD140AabRT   Price: $312.00/vial

Package Size: 100 micrograms in 1ml 10mM PBS pH 7.2 with   150mM NaCl and with 0.09% NaN3 preservative

Clone: APA5

Ig Isotype: rat IgG2ak


Antigen: mouse PDGF receptor alpha chain-human IgG1 recombinant fusion protein

Purification: Protein G column from tissue culture supernatant

Reactivity: Reacts with murine PDGF Receptor alpha chain which is widely expressed on cells of mesenchymal origin in the embryo and adult and on several other cell types during embryonic development, but not on hematopoietic cells. PDGFR-A binds to PDGF A and B chains, in contrast to PDGFR-B which binds only to the PDGF B chain. Biologically active PDGF is a disulphide-linked dimer, forming the AA, AB and BB isoforms . This monoclonal has been demonstrated on block binding of PDGF-AA to PDGF-alpha expressing cells in vitro and to block some PDGF- mediated development events in vivo.

Uses: -immunofluorescent staining and flow cytometry (approx 1ug/million cells)

           -histochemistry (acetone fixed frozen sections 0.5-2.5ug/ml) & PARAFFIN SECTIONS REF#1

            -WESTERN BLOT (SEE REF#1)

            -elisa

              -blocking experiments (with azide free material) (availabel on special order only, cat#RDI-MCD140abrt-2X $3125.00/2mg minimum order

Storage: Store at 4 Deg C.

REF: -J. Invest Dermatol. 107:770-777

         -Neuron 17:1117-1131

       -EMBO J. 11:4251-4259

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics. Sodium azide may form explosive compounds in presence of heavy metals or under acidic conditions. Flush drains with copious amounts of water to prevent buildup of explosive compounds. 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.


Rabbit anti-PDGFR-Alpha

cat# RDI-PDGFRAabrX   $500.00

Presentation: 200ul immunoaffinity purified rabbit IgG in 0.2M Tris, 0.1mM EDTA, 150mM Nacl, 45mM glycine, 5mg/ml BSA, 0.05% sodium azide and 30% glycerol

Reactivity: reacts with human and mouse (predicted to react with rat, chicken, frog and zebra fish based on sequence homology

immunogen: KLH conjugated peptide aa1035-1053 (C-GKRNHSSQTSEESAIETG) from human PDGFR alpha, Peptide has 19/19 idential amino   acid in mouse, chicken, rat and frog, and 1819 in zebra   fish

use: -western blot: 1:250-1:1000 dilution recognizes PDGFRA as doublet as 150/170kda, pos control 3T3/NIH cell lysate

-immunoprecipiation: 5-10ul immunoprecipiated PDGFR-A from 500ug of 3T3/NIH RIPA lysate

-no tested for other applications

Storage: Stable for 1 year at -20°C from date of shipment. For maximum recovery of product, centrifuge the vial prior to removing the cap.

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

sample Immunoblot Protocol

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate sample (cell lysis buffer: 50mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150mM NaCl; 1mM EGTA; 1mM PMSF; 1ug/ml each aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.

2. Block the blotted nitrocellulose in freshly prepared 3% nonfat dry milk in TBS (TBS-MLK) for 30 minutes at room temperature with constant agitation.

3. Incubate the nitrocellulose with a 1:500 dilution of anti-PDGF Receptor a, diluted in freshly prepared TBS-MLK overnight with agitation at 4°C.

4. Wash the nitrocellulose twice with water.

5. Incubate the nitrocellulose in the secondary reagent of choice (an anti-rabbit HRP conjugated IgG, in TBS-MLK for 1.5 hours at room temperature with agitation.

6. Wash the nitrocellulose with water twice.

7. Wash the nitrocellulose in TBS-0.05% Tween 20 for 3-5 minutes.

8. Rinse the nitrocellulose in 4-5 changes of water.

9. Use detection method of choice (enhanced chemiluminescence was used).

Immunoprecipitation Protocol

1. Dilute the cell lysate before beginning the immunoprecipitation to roughly 1ug/ul total cell protein in a microcentrifuge tube with modified RIPA lysis buffer plus inhibitors.

2. Add 5ul of anti-PDGF Receptor a to 500ug cell lysate.

3. Gently rock the reaction mixture at 4°C for 2 hours.

4. Capture the immunocomplex by adding 100ml (50ul packed beads) of washed Protein A agarose bead slurry

5. Gently rock the reaction mixture at 4°C for 1 hour.

6. Collect the agarose beads by pulsing (5 seconds in the microcentrifuge at 14,000 x g), and drain off the

supernatant. Wash the beads 3 times with either ice-cold cell lysis buffer or PBS.

7. Suspend the agarose beads in 60ml 2X Laemmli sample buffer.

8. Store the beads frozen for future analysis or boil the beads for 5 minutes.

9. Collect the beads after boiling using a microcentrifuge pulse.

10. Perform SDS-PAGE and immunoblot analysis on a sample of the supernatant fraction.

For Research Use Only


Rabbit anti-PDGFR-BETA

cat# RDI-PDGFRBabrX   $500.00

Presentation: 200ug protein A purified rabbit IgG in 200ul of 0.1M Tris-glycine buffer, pH 7.4, 0.15M NaCl, containing 0.05% sodium azide.

Reactivity: reacts with human and mouse (others not tested)

immunogen: Synthetic peptide (QPNEGDNDYIIPLA) corresponding to the aa 1013-1025 of Human PDGF Receptor Type B containing a carboxy-terminal alanine residue.

use: -western blot: 1-2ug/ml detected the PDGF Receptor Type B (~190kDa) in RIPA lysates from mouse 3T3/A31 fibroblasts The specificity of the immunoreaction was previously confirmed by using 1mM of the immunizing peptide to inhibit the antibody.

-Immunoprecipitation: 4ug of this lot immunoprecipitated the PDGF Type B Receptor from 500ug of 3T3/A31 RIPA lysate.

-Immunocytochemistry: 5-10ug/ml showed positive immunostaining for the PDGF Receptor Type B in 3T3/A31 cells fixed with 4% paraformaldehyde for 10 minutes.

-no tested for other applications

Storage: Stable for 1 year at -20°C from date of shipment. For maximum recovery of product, centrifuge the vial prior to removing the cap.

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

sample Protocol

Immunoprecipitation Protocol

1. Before beginning the immunoprecipitation, dilute the cell lysate to roughly 1ug/ul total cell protein in a microcentrifuge tube with PBS.

2. Add 4µg of anti-PDGF Receptor Type B to 500ug-1mg cell lysate.

3. Gently rock the reaction mixture at 4° C overnight.

4. Capture the immunocomplex by adding 100ul (50ul packed beads) of washed Protein A agarose bead slurry.

5. Gently rock the reaction mixture at 4° C for 2 hours.

6. Collect the agarose beads by pulsing (5 seconds in the microcentrifuge at 14,000 x g), and drain off the supernatant. Wash the beads 3 times with either ice-cold cell lysis buffer or PBS.

7. Resuspend the agarose beads in 50ml 2X Laemmli sample buffer.

8. The agarose beads can either be frozen for later use or suspended in Laemmli sample buffer and boiled for 5 minutes. The beads are collected by a microcentrifuge pulse and SDS-PAGE and subsequent immunoblot analysis performed on a sample of the supernatant.

Western Immunoblot Protocol

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate sample (cell lysis buffer: 50mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150mM NaCl; 1mM EGTA; 1mM PMSF; 1mg/ml aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.

2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 20 minutes at 20-25° C with constant agitation.

3. Incubate the nitrocellulose with 0.5-2µg/ml of anti-PDGF Receptor Type

B, diluted in freshly prepared PBS-MLK overnight with agitation at 4° C.

4. Wash the nitrocellulose twice with water.

5. Incubate the nitrocellulose in the secondary reagent of choice (an anti-rabbit IgG linked to horseradish peroxidase, was used) in PBS-MLK for 1.5 hours at room temperature with agitation.

6. Wash the nitrocellulose with water twice.

7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-5 minutes.

8. Rinse the nitrocellulose in 4-5 changes of water.

9. Use detection method of choice (enhanced chemiluminescence was used).

Immunocytochemistry

1. Plate approximately 200ul of cell suspension into each well of a slide. Incubate 24 hours in a 37° C CO2 incubator.

2. Wash the cells three times for 15 minutes with PBS. Do not shake cells.

3. Add fix (4% paraformaldehyde) in PBS for 10 minutes at room temperature.

4. Wash the cells with PBS, twice, for 15 minutes. Do not shake.

5. Add 400ml of 8% BSA in PBS and incubate for 1 hour at room temperature.

6. Wash the cells twice with PBS, for 15 minutes.

7. Incubate the cells with 10µg/ml anti-PDGF Receptor Type B in 1% BSA in PBS and incubate for 1 hour at room

temperature.

8. Wash the cells twice with PBS, for 15 minutes.

9. Incubate the cells with a 1:100 dilution of goat anti-rabbit IgG fluorescein conjugated secondary antibody in 1% BSA in PBS for 1 hour at room temperature.

10. Wash the cells three times with PBS, for 15 minutes.

11. Examine the cells under a fluorescent microscope

For Research Use Only


Rabbit anti-PHOSPHO-PDGFR-Beta

cat# RDI-PDGFRBPHabrX    $562.00

Presentation: 200ug of protein A purified rabbit IgG in 200ul of 0.1M   Tris-glycine, pH 7.4, 0.15M NaCl with 0.05% sodium azide   and 30% glycerol.

Reactivity: reacts with Mouse; predicted to cross react with human and rat based on conservation of immunogenic sequence.

immunogen: KLH-conjugated, synthetic peptide corresponding to amino acids 710-718 of human PDGF Receptor b (C-PPSAELpYSN). An   N-terminus cysteine was added to facilitate conjugation.   This sequence corresponds to amino acids 709-717 of   mouse PDGFR b.

Specificity: Recognizes PDGFR b phosphorylated at Y716, Mr 190kDa; an   unknown protein was also recognized, Mr 66kDa. Shows   cross-reactivity with the phosphorylated EGF receptor.

use: -Immunoblot Analysis: 4ug/ml detected PDGFR b phosphorylated at Y716 in RIPA lysates from PDGF-stimulated mouse 3T3 cells. Note: Immunoprecipitation of the PDGF receptor using anti-PDGFR is recommended to help discriminate   between the PDGFR and EGFR.

-not tested for other applications

Storage: Store at -20°C .Avoid frequent freeze thaw cycles. Recommend aliquoting and store -20 DEG C. For maximum recovery of product,   centrifuge the vial prior to removing the cap.

Immunoblot Protocol

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a lysate from PDGF-stimulated cells (cell lysis buffer: 50mM Tris-HCl, pH7.4; 1% NP-40; 0.25% sodium deoxycholate; 150mM NaCl; 1mM EGTA; 1mM PMSF; 1ug/ml each aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.

2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 60 minutes at 20-25°C with constant agitation.

3. Incubate the nitrocellulose with 4ug/ml of anti-phospho-PDGFR b (Y716), diluted in freshly prepared PBS-MLK overnight with agitation at 4°C.

4. Wash the nitrocellulose twice with water.

5. Incubate the nitrocellulose in the secondary reagent of choice (an anti-rabbit HRP conjugated IgG was used) in PBS-MLK for 45 minutes at room temperature with agitation.

6. Wash the nitrocellulose with water twice.

7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-5 minutes.

8. Rinse the nitrocellulose in 4-5 changes of water.

9. Use detection method of choice (enhanced chemiluminescence was used).

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


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RDI Divison of Fitzgerald Industries Intl

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