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9386
Phospho-Threonine (42H4) Mouse mAb
Primary Antibodies
Monoclonal Antibody

Phospho-Threonine (42H4) Mouse mAb #9386

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Citations (31)
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  1. WB
  2. IP
Western Blotting Image 1: Phospho-Threonine (42H4) Mouse mAb

Western blot analysis of extracts from A431 cells, untreated or treated with calyculin A, a threonine phosphatase inhibitor, or extracts from NIH/3T3 cells, untreated or treated with pervanadate, a tyrosine phosphatase inhibitor, using Phospho-Threonine (42H4) Mouse mAb (upper) or Phospho-Tyrosine Monoclonal Antibody (P-Tyr-100) #9411 (lower).

No image available
Product Image 1: Phospho-Threonine (42H4) Mouse mAb

Phospho-Threonine (42H4) Mouse mAb ELISA Assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (T* denotes phosphorylated threonine.)

To Purchase # 9386S
Product # Size Price
9386T
20 µl $ 126
9386S
100 µl $ 258

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgM

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Peptide ELISA (DELFIA) 1:2000

Storage

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.

Protocol

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Western Blotting Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-mouse IgG, HRP-linked Antibody (#7076 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 262

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for western immunoblot analysis or kinase activity assays.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723). Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein L Agarose Beads: Use Protein L for mouse IgM immunoprecipitation.
  5. 10X Kinase Buffer (for kinase assays): (#9802) To prepare 1 ml of 1X kinase buffer, add 100 μl 10X kinase buffer to 900 μl dH2O, mix.
  6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 μM), add 10 μl ATP (10 mM) to 490 μl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 μl of 50% Protein L agarose bead slurry to 200 μl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 μl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add protein L agarose (10–30 μl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 μl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 μl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 μl 1X kinase buffer supplemented with 200 μM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 μl) on a 4–20% gel for SDS-PAGE.

posted January 2016

Protocol Id: 884

ELISA Peptide

A. Solutions and Reagents

  1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3 (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer.
  2. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  3. Wash Buffer: 1X PBS containing 0.05% Tween-20 (PBST)
  4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
  5. Antibody Dilution Buffer: 3% BSA in PBST
  6. DELFIA® Europium-labeled Anti-mouse IgG for mouse primary antibodies or Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124) for rabbit primary antibodies.
  7. DELFIA® Enhancement Solution (PerkinElmer Life Sciences #1244-105)

(DELFIA® is a registered trademark of PerkinElmer, Inc.)

B. Protocol

  1. Coat the wells of a 96-well microtiter plate with 100 μl of 1 μM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hrs at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
  2. Wash plate three times 200 μl/well with wash buffer.
  3. Block plate with 200 μl/well blocking buffer for 1 hr at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)
  4. Prepare appropriate dilution of primary antibody with antibody dilution buffer. Add 100 μl to wells and incubate at 37°C for 1 hr.
  5. Wash three times with wash buffer.
  6. Add 67 ng/well DELFIA Europium-labeled Anti-mouse IgG or Anti-rabbit IgG, diluted in 100 μl/well antibody dilution buffer. Incubate at room temperature for 30 min, on gentle shaker.
  7. Wash three times with wash buffer.
  8. Add 100 μl enhancement solution and incubate at room temperature for 5 min. Read plate at 615 nm with an appropriate time-resolved plate reader.

posted June 2005

revised September 2007

Protocol Id: 34

Specificity / Sensitivity

Phospho-Threonine (42H4) Mouse mAb binds phosphorylated threonine residues in a manner largely independent of the surrounding amino acid sequence. The antibody is phospho-specific but does not cross-react with phospho-tyrosine-containing sequences. It does show slight cross-reactivity with a few phospho-serine-containing peptides. By ELISA, it recognizes a wide variety of threonine-phosphorylated peptides. (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.)

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with phospho-Thr-containing peptides.

Background

Much of the dynamic behavior of cellular proteins, including the regulation of molecular interactions (1), subcellular localization (2), and transcriptional regulation (3) is controlled by a variety of post-translational modifications (4). Antibodies specific for these post-translational modifications are invaluable tools in the quest to understand normal and pathogenic molecular and cellular behavior.

General protein modification antibodies are designed to react with modified amino acid residues (e.g. phospho-threonine, phospho-tyrosine, acetyl-lysine, nitro-tyrosine) independently of the sequence in which they are embedded. This ability to recognize modified residues in a "context-independent" fashion gives these antibodies broad reactivities, presumably conferring upon them the ability to react with hundreds of distinct proteins. This broad pattern of reactivity makes these antibodies especially valuable in multiplex analyses and target discovery programs.

Protein kinases are among the most abundant eukaryotic regulatory proteins; over 500 separate kinase genes are encoded in mammalian genomes (5,6). In spite of the importance of kinases in eukaryotic biology, relatively few of their physiological targets are known. Phospho-Threonine Antibody (P-Thr-Polyclonal) #9381 and Phospho-Threonine (42H4) Monoclonal Antibody #9386 provide powerful tools for discovering targets of serine/threonine kinases, for monitoring and characterizing in vitro threonine phosphorylation reactions as well as for high throughput Ser/Thr kinase drug discovery.

  1. Yaffe, M.B. and Elia, A.E. (2001) Curr Opin Cell Biol 13, 131-8.
  2. Appella, E. and Anderson, C.W. (2001) Eur J Biochem 268, 2764-72.
  3. Jenuwein, T. and Allis, C.D. (2001) Science 293, 1074-80.
  4. Krishna, R.G. and Wold, F. (1993) Adv Enzymol Relat Areas Mol Biol 67, 265-98.
  5. Venter, J.C. et al. (2001) Science 291, 1304-51.
  6. Manning, G. et al. (2002) Science 298, 1912-34.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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 [email protected] For information regarding commercial licensing terms please contact CST Pharma Services Department at [email protected]