rev: May 5, 2003

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ANTIBODIES  

(anti-Human and others as indicated)

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (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.


Anti-Phosphothreonine ab  

Other Phosphorylation Antibodies: (BULK QUOTES ON REQUEST)

#RDI-PHOSTYRabr   rabbit anti-Phosphotyrosine     50ug $190.00

#RDI-PHOSSERabr   rabbit anti-Phosphoserine        50ug $190.00

#RDI-PHOSTHRabr   rabbit anti-phosphothreonine   50ug $190.00

#RDI-PHOSTPabr      rabbit anti-Phosphothreonine-Proline 50ug   $190.00

#RDI-PHOSTLYTabr  rabbit anti-Phosphothreonine-Lysine-phosphotyrosine 50ug  $190.00


Rabbit anti-phosphothreonine

cat#RDI-PHOSTHRabr    $188.00/vial 50ug    Bulk quotes on request

also available Biotin conjugated: cat#RDI-PHOSTHRR-BT  $250.00/50ug

-affinity purified rabbit antibody made against phosphothreonine containing proteins-reacts specifically with threonine phosphorylated proteins and shows no reactivity to either phosphoserine or phosphotyrosine.A431 cells. -suitable for western blot, immunoprecipitation and Elisa

Sample Product Specs:

Product: Affinity Isolated Rabbit Anti-Phosphothreonine Antibodies (anti-pT)

Catalog: RDI-PHOSTHRabr  $188.00/50ug  

-also available Biotin labeled: cat#RDI-PHOSTHRR-BT $250.00/50ug

Immunogen: KLH-phosphothreonine conjugates

Purification: Immunoaffinity chromatography with phosphothreonine-agarose

Presentation: 50ug in 0.125ml (400ug/ml) in PBS with 0.05% NaN3

Strorage: at 4 DEG C, do not freeze

Specificity & Reactivity: Both antigen-capture and antibody-capture ELISA indicated that the anti-phosphothreonine antibodies can recognize threonine-phosphorylated protein, phosphothreonine and lysine-phosphothreonine-glysine random polymer, respectively. Direct, competitive antigen-capture ELISA demonstrated that the antibodies are specifically inhibited by free phosphothreonine, phosvitin but not by free phosphoserine, phosphotyrosine, threonine and ATP.

Sensitivity: Detecting 100 ng of phosvitin and active ERK1 with immunoblotting, 0.5 ng of phosvitin with ELISA



Applications: ELISA (kinase assay), 0.5ug/mL; Western blot (0.5ug/ml if using non chemiluminescence detection system, if using chemiluminescence, recommend tittering from 1-0.4ug/ml) , 4ug/mL; IP, 10ug/500ug protein sample

USE: non-radioactive protein kinase assay (ELISA) using biotinylated peptide substrate; immunoblotting of BSA-peptide substrate conjugates phosphorylated by kinase; Immunoblotting of abundant phosphoprotein. Not recommend for immunoblotting of trace cellular phosphoprotein (enhanced chemiluminescence detection may be required). Acetone precipitation of the protein extract followed by SDS denaturation is recommended for successful immunoprecipitation.

Positive Ctrl: Mouse brain extract for immunoblotting. Phosvitin for ELISA

Note: For research use only.


Sample Immunoprecipitation Protocol:

Rabbit anti-Phosphoserine (pS) and rabbit anti-Phosphthreonine (pT) antibodies

The positive control for IP:

anti-pS: phosphorylated tau protein in brain sample, biotinylated phosvitin

anti-pT: active ERK1/2 (44/42 Kd), CamK (56, 46 Kd) in the brain sample.



Procedure Immunoprecipitation of Phosphoprotein with Affinity Purified Anti-pS and Anti-pT

1. Materials: affinity isolated anti-pS and anti-pT (400 ug/mL in PBS); 50mM Tris (pH.8.5); beta glycerolphosphate (Sigma cat#G6251, phosphatase inhibitor)); IP buffer (50 mM Tris, 0.05% NP40, 100 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 ug/mL of aprotonin, pepstatin and leupeptin, 1 mM NaF, final pH 7.5); protein A agarose (blocked with 5% BSA)

2. Preparation of the mouse brain extract.

Cut 0.5 gram of mouse brain sample into small pieces in 5 mL of the IP buffer plus 10 mg of beta glycerolphosphate (2mg/ml in final concentration). Homogenize  the tissue at 20,000 rpm for 5-10 seconds. Repeat the homogenization several times 5 min  interval. Keep the tissue on ice all the time.

a) Transfer the homogensate to a 10 mL centrifuge tube and centrifuge at15,000 rpm at 4 DEG C for 45 min.

b) Using a needle to puncture the bottom of the tube (above the pellet) and collect 2.5 mL of the supernatant. Avoid the collection of the fat layer on top of the tube. Check protein concentration (should be greater than 5 mg/mL)

c) Acidify the extract with 25 uL of the concentrated phosphoric acid.Allow the inactivation of the enzyme for 2 hr.

d) Add at least five volume of acetone to precipitate the proteins in the extract.

e) Centrifuge the acetone mixture at 5000 rpm at 4 DEG C. Discard the solution and wash the pellet with acetone twice. Try to remove as much phospholipid and acid as possible using acetone.

f) Crunch the pellet with a pipette tip and reconstitute the pellet in 0.5 mL of distill water. Add 50 uL of the beta glycerolphosphate in 50 mM Tris buffer (pH8.5) and  heat (50-80oC) for 10 min.

g) Centrifuge the mixture at 15,000 rpm for 30 min. Collect the supernatant for IP or store at -20oC for future use.

3. Immunoprecipitation
a) Take 100 uL approximately 500 ug total brain proteins into a microcentrifuge tube. Check final pH (7.5-8.0) or adjust pH with phosphoric acid and Tris. Add 5 uL of isopropanol in the tube.

b) Add 20 ug of anti-pT or anti-pS in the tube, vortex. Allow the bind at 4-8 DEG C overnight.

c) Add 20 uL of protein A agarose to the tube and adjust the final volume to 500 uL with IP buffer

d) Incubate the mixture in a rotary rack for 2 hrs.

e) Wash the protein A beads with IP buffer 4 times.

f) Add 50-100 uL 4% SDS sample loading buffer and heat at 50-80 DEG C for 2- 5 min.

h) Load the slurry to SDS-PAGE for western blot analysis


Mouse anti-Phosphothreonine

cat#RDI-PHOSTHabm   $688.00/vial 0.5ml

-clone ref# PTR-8

-mouse IgG2b

--as determined in Elisa and immunoblot, the antibody reacts specifically with phosphorylated threonine, both as free amino acid or conjugated to carriers as BSA or KLH. No cross reactivity is observed with non-phosphorylated threonine, phophoserine, phosphotyrosine, AmpMP or ATP.

-can be used in immunoblotting for the localization of some phosphosthreonine-containing proteins. Certain proteins known to contain phosphorylated threonine may not be recognized by this antibody due to steric hindrance at the recognition site.

-approx use dilution of 1:50 by immunoblotting and 1:2000 in indirect Elisa.

-also available biotin labelled, cat#RDI-PHOSTHabm-Bt   $625.00/0.5ml

-negative inhibitory control cat#RDI-PHOSTH-BSA  $125.00/0.2ml


All products are for in vitro research use only-Not for use in or on humans or animals-Not for Diagnostic use. Not responsible for any patent infringements with the use or derivation of these products. Price/specifications/availability subject to change without notice.


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RDI Division 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

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