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5286
Phospho-Bad (Ser136) (185D10) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Phospho-Bad (Ser136) (185D10) Rabbit mAb #5286

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Citations (5)
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  1. WB
Western Blotting Image 1 - Phospho-Bad (Ser136) (185D10) Rabbit mAb

Western blot analysis of extracts from control 293, GST-Bad transfected 293 and COS cells, untreated or TPA-treated, using Phospho-Bad (Ser136) (185D10) Rabbit mAb.

To Purchase # 5286S
Product # Size Price
5286T
20 µl N/A
5286S
200 µl N/A

Supporting Data

REACTIVITY M Mk
SENSITIVITY Transfected Only
MW (kDa) 23
Source/Isotype Rabbit IgG

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 1:500

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

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

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-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 (#13953, 5 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-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 June 2020

Protocol Id: 10

Specificity / Sensitivity

Phospho-Bad (Ser136) (185D10) Rabbit mAb detects transfected levels of Bad protein only when phosphorylated at Ser136.

Species Reactivity:

Mouse, Monkey

Species predicted to react based on 100% sequence homology:

Human, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser136 of mouse Bad.

Background

Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xL (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation at Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).

  1. Yang, E. et al. (1995) Cell 80, 285-291.
  2. Zha, J. et al. (1996) Cell 87, 619-628.
  3. Datta, S.R. et al. (1997) Cell 91, 231-241.
  4. Peso, L. et al. (1997) Science 278, 687-689.
  5. Bonni, A. et al. (1999) Science 286, 1358-1362.
  6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
  7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
  8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
  10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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