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.


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-Phosphoserine

cat#RDI-PHOSSERabr   $188.00/50ug vial  (BULK MG Quoted on request)

-also available Biotin conjugated  cat#RDI-PHOSSERR-BT  $250.00/50ug   $219.00/vial 3 or more

-affinity purifed rabbit antibody made against phosphoserine containing proteins

-specificity: reacts specifically with serine phosphorylated proteins and shows no significant cross reactivity to other phosphothreonine or phosphotyrosine by western blot analysis.

-suitable for western blot, immunoprecipitation and ELISA.

Sample Data Sheet

Product: Affinity Isolated Rabbit Anti-Phosphoserine Antibodies (anti-pS)

Catalog: RDI-PHOSSERabR  $188.00/50ug
       -also available Biotin conjugated: cat#RDI-PHOSSERR-BT   $250.00/50ug

Immunogen: KLH-phosphoserine conjugates

Purification: Immunoaffinity chromatography with phosphoserine-agarose

Presentation: 50ug in 0.125ml (400ug/mL) in PBS with 0.02% NaN3

Storage: at 4 DEG C, do not freeze

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

Sensitivity: Detecting 50 ng of phosvitin with immunoblotting, 0.5 ng of phosvitin or 5 ng of a-casein with ELISA

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

Recommendation: non-radioactive protein kinase assay (ELISA) using bi0tinylated peptide substrate, immunoblot of BSA-peptide substrate conjugates phosphorylated by a kinase. Immunoblotting of abundant phosphoprotein. Not recommend for immunoblotting of trace cellular phosphoprotein (enhanced chemiluminescence detection required). Acetone precipitation of the protein extract followed by SDS denaturation is recommended for successful immunoprecipitation

Positive Control: Mouse brain extract or phoshorylase a for immunoblotting. Phosvitin or a-casein for ELISA

Note: For research use only

1. Specificity as determined by direct, antigen-capture, competitive ELISA:

     Immobilized Ab: anti-pS (.5 mg/100 mL/well in PBS) onto 96-well microplate, physical adsorption overnight.

     Constant antigen:Ly-pS-Gly-HRP (random polymer) conjugates (0.2 mg/100 mL/well in TBSt with 0.5% BSA)

                                   % inhibiton of binding of Ly-pS-Gly-HRP to anti-pS  
Competitive antigens 95%  50% 10%
Phosphoserine 100 ug/mL 10-25 ug/mL 1-5 ug/mL
Phosphothreonine NA up to 1mg/mL 500 ug/mL 100 ug/mL
Phosphotyrosine no apparent inhibition up to 1 mg/mL
L-Serine no apparent inhibition up to 1 mg/mL

2.  Specificity as determined by indirect, antibody-capture ELISA:

Immobilized Antigens: Phosvitin (pS containing protein), Phosphothreonine (pT)-Glycine-Lysine random polymer and Phosphotyrosine (pY)-Lysine-Glycine random polymer

    Incubation: 10 ug/mL in PBS at 39oC for 2 hrs

    Blocking: 2% BSA at room temp for 30 min

1st Antibodies: anti-pS starting from 0.3 ug/mL in TBSt with 0.5% BSA followed by 2x serial dilution.

Incubation: at room temp for 30 min.

2nd Antibody: goat anti-rabbit Ig HRP conjugates (1 mg/mL in TBSt with 0.5%cBSA)

Incubation: at room temp for 30 min

Figure: Binding of anti-pS to different immobilized antigen: phosvitin, Ly-pT-Gly random polymer and Ly-pY-Gly random polymer.

Immunoblotting

Figure 1. Sensitivity of detection of a known phosphoprotein in western blotting system using with anti-pS (cat#RDI-PHOSSERabr)

A      3 ug/lane

B     1 ug/lane

C    0.3 ug/lane

D     0.1 ug/lane

Primary antibody: anti-pS (4 ug/mL in TBSt) at room temperature for 4 hrs.

Secondary: goat anti-rabbit Ig ALP conjugates (1 mg/mL in TBSt) at room temperature for 1 h.   (or -HRP conjugates with chemiluminescence detection)

Figure 2. Western Blotting Mouse Brain Extract with Affinity Purified Anti-phosphoserine

A. 125 ug

B. 75 ug

C. 25 ug

D. 10 ug

Primary antibody: anti-pS (4 ug/mL in TBSt) at room temperature for 4 hrs.

Secondary: goat anti-rabbit IgG ALP conjugates (1 ug/mL in TBSt) at room temperature for 1 h.

Note: (a blocking solution containing 2-3% BSA is recommended). Skim Milk should be avoided as it can contain phosphoproteins and interfere with antibody binding.


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-Phosphoserine

cat#RDI-PHOSSEabm   $688.00/vial 0.5ml

-clone ref#PSR-45

-mouse IgG1

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

-can be used in immunoblotting (Abu-Lawi, K.I. et al, Infect.immunol., 63: 498 (1995) for the localization of some phosphoserine-containing proteins. Certain proteins known to contain phosphorylated serine may not be recognized by this antibody due to steric hindrance at the recognition site.

-approx use dilution of 1:50 by immunoblotting and 1:4000 in indirect Elisa.

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



Rabbit anti-Phosphoserine (BULK Material/ELISA)

cat# #RDI-PHOSSERabrX

Price: $938.00/1mg    $750.00/mg 10-24mg     $625.00/mg 25-49mg      $500.00/mg 50 or more

packaging: liquid IgG fraction in 10mM PBS with 150mM   NaCl, pH 7.2 & 0.1% NaN3,

Species: Rabbit

Purification: Ion Exchange chromatography

Activity: -to be determined, for bulk assay use

Storage: At 2-8 DEG C (short term) or aliquot and store -20 DEG C (long term). Avoid frequent freeze thaw cycles.

For In Vitro Research Use Only-Not For Use IN Or On Humans or Animals-Not For Use in Diagnostics.


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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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