ただ今実施中のプロモーション | 詳細はこちら >>
47477
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate) #47477

Reviews ()
Citations (0)
Filter:
  1. IF

Confocal immunofluorescent analysis of L-929 (NCTC clone 929) cells, untreated (left) or pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 2 hr; right), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

To Purchase # 47477S
製品番号 サイズ 価格 在庫
47477S
100 µl  (50 tests)

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
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 Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct immunofluorescent analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb #91702.

Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:200

Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

PRINT

View >Collapse >

Immunofluorescence (Immunocytochemistry)

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 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 182

Specificity / Sensitivity

Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of RIP3 protein only when phosphorylated at Thr231/Ser232.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Thr231/Ser232 of mouse RIP3 protein. This antibody may not recognize RIP3 when only singly phosphorylated at Thr231 or Ser232.

Background

The receptor-interacting protein (RIP) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that trigger pro-survival and inflammatory responses through the activation of NF-κB, as well as pro-apoptotic pathways (1). In addition to the kinase domain, RIP contains a death domain responsible for interaction with the death domain receptor Fas and recruitment to TNF-R1 through interaction with TRADD (2,3). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (4,5). RIP also interacts with TNF-receptor-associated factors (TRAFs) and can recruit IKKs to the TNF-R1 signaling complex via interaction with NEMO, leading to IκB phosphorylation and degradation (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (2,3). Caspase-8-dependent cleavage of the RIP death domain can trigger the apoptotic activity of RIP (8).

  1. Meylan, E. and Tschopp, J. (2005) Trends Biochem Sci 30, 151-9.
  2. Hsu, H. et al. (1996) Immunity 4, 387-96.
  3. Stanger, B.Z. et al. (1995) Cell 81, 513-23.
  4. Ting, A.T. et al. (1996) EMBO J 15, 6189-96.
  5. Kelliher, M.A. et al. (1998) Immunity 8, 297-303.
  6. Devin, A. et al. (2000) Immunity 12, 419-29.
  7. Zhang, S.Q. et al. (2000) Immunity 12, 301-11.
  8. Lin, Y. et al. (1999) Genes Dev 13, 2514-26.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
ProLong is a registered trademark of Life Technologies Corporation.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

Powered By OneLink