Buy Three, Get the Fourth Free! | Start Saving >>
23640
ASC (D2W8U) Rabbit mAb (Mouse Specific) (Alexa Fluor® 647 Conjugate)
Antibody Conjugates

ASC (D2W8U) Rabbit mAb (Mouse Specific) (Alexa Fluor® 647 Conjugate) #23640

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
IF-IC

Confocal immunofluorescent analysis of mouse primary bone marrow-derived macrophages differentiated with mM-CSF (20 ng/ml, 8 d), either treated with Lipopolysaccharides (LPS) #14011 (50 ng/mL, overnight; left) or LPS (50 ng/mL, overnight) followed by Nigericin (10 μM, 2 hr; right), using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (Alexa Fluor® 647 Conjugate) (green pseudocolor). Blue = Hoechst 33342 #4082 (fluorescent DNA dye). Note the translocation of ASC to inflammasomes following stimulation with LPS and Nigericin (white arrow).

Learn more about how we get our images.

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

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:50
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.

ASC (D2W8U) Rabbit mAb (Mouse Specific) (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total ASC protein.

Species Reactivity:

Mouse

Monoclonal antibody is produced by immunizing animals with recombinant mouse ASC protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct immunofluorescent analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated ASC (D2W8U) Rabbit mAb (Mouse Specific) #67824.

TMS1 (target of methylation-induced silencing)/ASC (apoptosis-associated speck-like protein containing a CARD), also referred to as PYCARD and CARD5, is a 22-kDa pro-apoptotic protein containing an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD) (1-2). The ASC/TMS1 gene was originally found to be aberrantly methylated and silenced in breast cancer cells (2), and has since been found to be silenced in a number of other cancers, including ovarian cancer (3), glioblastoma (4), melanoma (5), gastric cancer (6), lung cancer (7), and prostate cancer (8). Expression of ASC/TMS1 can be induced by pro-apoptotic/inflammatory stimuli (9). During apoptosis ASC/TMS1 is re-distributed from the cytosol to the mitochondria and associates with mitochondrial Bax to trigger cytochrome c release and subsequent apoptosis (10). ASC/TMS1 has also been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (11).

  1. Masumoto, J. et al. (1999) J Biol Chem 274, 33835-8.
  2. Conway, K.E. et al. (2000) Cancer Res 60, 6236-42.
  3. Terasawa, K. et al. (2004) Clin Cancer Res 10, 2000-6.
  4. Stone, A.R. et al. (2004) Am J Pathol 165, 1151-61.
  5. Guan, X. et al. (2003) Int J Cancer 107, 202-8.
  6. Moriai, R. et al. (2002) Anticancer Res 22, 4163-8.
  7. Virmani, A. et al. (2003) Int J Cancer 106, 198-204.
  8. Das, P.M. et al. (2006) Mol Cancer 5, 28.
  9. Strong, R. et al. (1991) Brain Res 542, 23-8.
  10. Ohtsuka, T. et al. (2004) Nat Cell Biol 6, 121-8.
  11. Srinivasula, S.M. et al. (2002) J Biol Chem 277, 21119-22.
Entrez-Gene Id
66824
Swiss-Prot Acc.
Q9EPB4
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 23640S

View sizes
Product # Size Price
23640S
100 µl  (50 tests) $ 299.0