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8767
CCN3 Antibody
一抗
多克隆抗体

CCN3 Antibody #8767

Citations (3)
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  1. WB
Western blot analysis of extracts from A172 and ACHN cells using CCN3 Antibody.
To Purchase # 8767S
Cat. # Size Price Inventory
8767S
100 µl

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 43
SOURCE Rabbit

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • 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
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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 nonfat dry milk, 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

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. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. 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 (#59329, 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 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: 263

Specificity / Sensitivity

CCN3 Antibody recognizes endogenous levels of total CCN3 protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg118 of human CCN3 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Background

CCN3, also named NOV (Nephroblastoma overexressed), belongs to the CCN (Cyr61, Ctgf, NOV) family of proteins. It is a cystine-rich secretory protein that associates with components of the extracellular matrix. Like other CCN family members, CCN3 is capable of mediating diverse biological functions through its four distinct domains, which enable binding to numerous protein partners (1-5).

CCN3 modulates bone turnover through various mechanisms and is implicated in the progression of primary bone cancers such as osteosarcoma and chondrosarcoma (6-8). Research has shown that CCN3 is also involved in the bone metastasis of melanoma, breast cancer, and prostate cancers (9-11). Recently, CCN3 was reported to play an important role in stem cell renewal (12). CCN3 is normally expressed in both embryonic and adult tissues (13,14). The activity of CCN3 is influenced by post translational modifications and proteolytic cleavage (15,16).

  1. Perbal, B. (2001) Mol Pathol 54, 57-79.
  2. Brigstock, D.R. et al. (2003) Mol Pathol 56, 127-8.
  3. Leask, A. and Abraham, D.J. (2006) J Cell Sci 119, 4803-10.
  4. Yeger, H. and Perbal, B. (2007) J Cell Commun Signal 1, 159-64.
  5. McCallum, L. and Irvine, A.E. (2009) Blood Rev 23, 79-85.
  6. Perbal, B. et al. (2008) Clin Cancer Res 14, 701-9.
  7. Tzeng, H.E. et al. (2011) J Cell Physiol 226, 3181-9.
  8. Yang, W. et al. (2011) Hum Reprod 26, 2850-60.
  9. Vallacchi, V. et al. (2008) Cancer Res 68, 715-23.
  10. Ouellet, V. et al. (2011) Am J Pathol 178, 2377-88.
  11. Chen, P.C. et al. (2012) Carcinogenesis 33, 937-45.
  12. Gupta, R. et al. (2007) Science 316, 590-3.
  13. Burren, C.P. et al. (1999) J Clin Endocrinol Metab 84, 1096-103.
  14. Kocialkowski, S. et al. (2001) Anat Embryol (Berl) 203, 417-27.
  15. Perbal, B. et al. (1999) Proc Natl Acad Sci U S A 96, 869-74.
  16. Su, B.Y. et al. (2001) Mol Pathol 54, 184-91.

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For Research Use Only. Not for Use in Diagnostic Procedures.
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