Western blot analysis of extracts from Jurkat cells, untreated or nocadazole-treated (1 µg/ml for 12 hours prior to lysis), using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101). Proteins were separated by 2D electrophoresis prior to blotting.
Western blot analysis of extracts from COS cells, untreated or serum and okadaic acid-treated, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (left). Right panel shows total protein staining.
Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma control (left) or lambda phosphatase-treated (right), using Phospho-Threonine-Prolin Mouse mAb (P-Thr-Pro-101).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing staining of proteins containing phospho-threonine-proline motifs, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101).
Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) ELISAs: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (T* denotes phosphorylated threonine.)
|Peptide ELISA (DELFIA)||1:1000|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 262
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted June 2005
revised March 2016
Protocol Id: 293
(DELFIA® is a registered trademark of PerkinElmer, Inc.)
posted June 2005
revised September 2007
Protocol Id: 34
Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) detects phospho-threonine only when followed by proline. It reacts with proteins and peptides phosphorylated at the Thr-Pro motif in an otherwise highly context-independent fashion. The antibody is phospho-specific, but does not recognize phospho-threonine in the absence of an adjacent proline. The antibody does not react with phospho-tyrosine but does react with some phospho-serine peptides containing the phospho-serine-proline motif (e.g., phospho-Elk-1). (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)
All Species Expected
Monoclonal antibody is produced by immunizing animals with synthetic phospho-threonine-proline-containing peptides. This antibody is a mouse IgM clone and can be recognized by anti-mouse Ig (whole molecule) secondary antibody.
The MAPK and CDK families of serine/threonine protein kinases play important roles in cell signaling and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (1-6). To facilitate the study and discovery of new MAPK and CDK substrates, Cell Signaling Technology has developed antibodies that bind to phospho-threonine or phospho-serine followed by proline.
As determined by ELISA using a wide variety of phospho-Thr-Pro peptides, Phospho-Threonine-Proline Monoclonal Antibody (P-Thr-Pro-101) recognizes the phospho-Thr-Pro motif in a highly context-independent fashion. It also interacts with a broad range of phospho-Thr-Pro-containing proteins as determined by western analysis of nocodazole-treated Jurkat cell extracts resolved on 2-D gels.
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