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REACTIVITY SENSITIVITY MW (kDa) Isotype
Endogenous Mouse 
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Immunohistochemical analysis of paraffin-embedded NCI-H460 (positive; left) or A431 (negative; right) cell pellets using DAX1 (D2F1) Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human testis using DAX1 (D2F1) Rabbit mAb.

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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 datasheet or 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 (Premixed Format): (#7720).
  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-rabbit IgG, HRP-linked Antibody (#7074).
  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 (#7720, 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 datasheet) 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-rabbit IgG, HRP-linked Antibody (#7074 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 November 2013

protocol id: 10

Western Blot Reprobing Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
  3. Deionized water (dH2O)
  4. Hematoxylin (optional)
  5. Wash Buffer:
    1X TBS/0.1% Tween-20 (1X TBST): To prepare 1 L add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.
    10X Tris Buffered Saline (TBS): To prepare 1 L add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl.
  6. *Antibody Diluent:
    1. SignalStain® Antibody Diluent #8112
    2. TBST/5% normal goat serum (#5425): To 5 ml 1X TBST add 250 µl normal goat serum.
    3. PBST/5% normal goat serum (#5425): To 5 ml 1X PBST add 250 µl normal goat serum.
      1X PBS/0.1% Tween-20 (1X PBST): To prepare 1 L add 100 ml 10X PBS to 900 ml dH2O. Add 1 ml Tween-20 and mix.
      10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phophate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  7. *Antigen Unmasking:
    1. Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
    2. EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
    3. TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1L add 1.21 g Trizma® base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 950 ml dH2O. Adjust pH to 9.0, then adjust final volume to 1000 ml with dH2O.
    4. Pepsin: 1 mg/ml in Tris-HCl pH 2.0.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
  10. Biotinylated secondary antibody.
  11. ABC Reagent: (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA) Prepare according to manufacturer’s instructions 30 minutes before use.
  12. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. *Antigen Unmasking

NOTE: Consult product data sheet for specific recommendation for the unmasking solution.

  1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
  2. For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 minutes at a sub-boiling temperature. No cooling is necessary.
  3. For TE: Bring slides to a boil in 10 mM TE/1 mM EDTA, pH 9.0 then maintain at a sub-boiling temperature for 18 minutes. Cool on the bench for 30 minutes.
  4. For Pepsin: Digest for 10 minutes at 37°C.

D. Staining

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.

NOTE: Consult product data sheet for recommended antibody diluent.

  1. Wash sections in wash buffer for 5 minutes.
  2. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
  3. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
  4. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
  5. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  6. If using ABC avidin/biotin method, prepare ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
  7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  8. Add 100-400 µl ABC reagent to each section and incubate for 30 minutes at room temperature.
  9. Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
  10. Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.
  11. As soon as the sections develop, immerse slides in dH2O.
  12. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  13. Wash sections in dH2O two times for 5 minutes each.
  14. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  15. Mount coverslips.

posted June 2005

revised February 2008

protocol id: 26

Product Usage Information

Application Dilutions
Western Blotting 1:5000
Immunohistochemistry 1:400
ELISA 1:1000

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.

Specificity / Sensitivity

Phospho-(Thr) MAPK/CDK Substrate Mouse mAb detects phospho-threonine only when followed by proline. It reacts with proteins/peptides phosphorylated on the Thr-Pro motif in an otherwise highly context-independent fashion. The antibody does not cross-react with phospho-threonine in the absence of an adjacent proline. The antibody does not cross-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.)


Source / Purification

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. Antibody is purified by protein A chromatography.

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 study and discover new MAPK and CDK substrates, Cell Signaling Technology has developed antibodies that bind to phospho-threonine followed by proline.


As determined by ELISA using a wide variety of phospho-Thr-Pro peptides, Phospho-(Thr) MAPK/CDK Substrate Monoclonal Antibody 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 blot analysis of nocodazole-treated Jurkat cell extracts resolved on a 2-D gel.


1.  Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol 200, 62-81.

2.  Seger, R. and Krebs, E.G. (1995) FASEB J 9, 726-35.

3.  Nurse, P. (2000) Cell 100, 71-8.

4.  Cross, T.G. et al. (2000) Exp Cell Res 256, 34-41.

5.  Yang, C.C. et al. (1998) J Protein Chem 17, 329-35.

6.  Reynolds, C.H. et al. (2000) J Neurochem 74, 1587-95.



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 CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at ptmscan@cellsignal.com.