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31174
TWIST1 (E7E2G) Rabbit mAb (IF Formulated)
Primary Antibodies

TWIST1 (E7E2G) Rabbit mAb (IF Formulated) #31174

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Confocal immunofluorescent tile scan of an E11.5 mouse embryo using TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Sample was mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue). Insets A – D are shown at higher resolution in subsequent figures.

Inset A from E11.5 mouse embryo stained with TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green), DyLight™ 554 Phalloidin #13054 (red), and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Inset B from E11.5 mouse embryo stained with TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green), DyLight™ 554 Phalloidin #13054 (red), and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Inset C from E11.5 mouse embryo stained with TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green), DyLight™ 554 Phalloidin #13054 (red), and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Inset D from E11.5 mouse embryo stained with TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green), DyLight™ 554 Phalloidin #13054 (red), and ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Confocal immunofluorescent analysis of a mouse digit from E14.5 embryo using TWIST1 (E7E2G) Rabbit mAb (IF Formulated). Transverse sections were labeled with TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (green) and DyLight™ 554 Phalloidin #13054 (red) before mounting in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Confocal immunofluorescent analysis of SH-SY5Y cells (left, positive) and HeLa cells (right, negative) using TWIST1 (E7E2G) Rabbit mAb (IF Formulated) (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 # 31174S
製品番号 サイズ 価格 在庫
31174S
100 µl

Supporting Data

REACTIVITY H M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Usage Information

Application Dilutions
Immunofluorescence (Frozen) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:400 - 1:1600

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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Immunofluorescence (Frozen)

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 serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. 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. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

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

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes 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. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. 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 July 2016

Protocol Id: 151

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

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 serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. 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. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. 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. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. 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: 24

Specificity / Sensitivity

TWIST1 (E7E2G) Rabbit mAb (IF Formulated) recognizes endogenous levels of total TWIST1 protein.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro24 of human TWIST1 protein.

Background

TWIST1 is a basic helix-loop-helix (b-HLH) transcription factor that functions as a master regulator of embryonic morphogenesis and plays essential roles in mesenchymal differentiation and osteogenic determination (1-3). Mutations affecting the b-HLH domain of the TWIST1 gene have been associated with Saethre-Chotzen syndrome, an autosomal dominant craniosynostosis disorder causing craniofacial and limb abnormalities (4,5). TWIST1 is upregulated in various human tumors and may play a role in EMT (epithelial-mesenchymal transition) and metastasis (6,7). Upregulation of TWIST1 may contribute to resistance to Taxol and microtubule regulating drugs in tumors (8).

  1. Thisse, B. et al. (1987) Nucleic Acids Res. 15, 3439-53.
  2. Lee, M.S. et al. (1999) J. Cell Biochem. 75, 566-77.
  3. Chen, Z.F. and Behringer, R.R. (1995) Genes Dev. 9, 686-99.
  4. Howard, T.D. et al. (1997) Nat. Genet. 15, 36-41.
  5. Connerney, J. et al. (2006) Dev. Dyn. 235, 1345-57.
  6. Watanabe, O. et al. (2004) Anticancer Res. 24, 3851-6.
  7. Yang, J. et al. (2004) Cell 117, 927-39.
  8. Wang, X. et al. (2004) Oncogene 23, 474-82.

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.

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