|H M R All||Endogenous||Mouse IgM|
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.Learn more about how we get our images.
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.Learn more about how we get our images.
Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101).Learn more about how we get our images.
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).Learn more about how we get our images.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101).Learn more about how we get our images.
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).Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
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
|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.
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.)
Human, Mouse, Rat, 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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.
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|9391S||100 µl||$ 303.0|