ADAMTS13 anticorps (ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 13) (AA 250-350)

Details for Product anti-ADAMTS13 Antibody No. ABIN152016
Antigène
  • ADAMTS13
  • ADAM-TS13
  • ADAMTS-13
  • C9orf8
  • VWFCP
  • vWF-CP
  • Gm710
  • ADAM metallopeptidase with thrombospondin type 1 motif 13
  • a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 13
  • ADAM metallopeptidase with thrombospondin type 1 motif, 13
  • ADAMTS13
  • Adamts13
  • adamts13
Épitope
AA 250-350
17
8
6
6
4
3
2
2
2
2
2
2
1
1
1
1
1
Reactivité
Humain
92
19
17
Hôte
Lapin
71
15
4
2
Clonalité
Polyclonal
Conjugué
Cet anticorp ADAMTS13 est non-conjugé
9
5
3
3
3
3
3
3
3
3
3
2
1
1
1
1
1
1
Application
Western Blotting (WB)
53
32
15
13
9
8
6
6
6
5
3
2
2
1
Options
Immunogène A synthetic peptide which maps to a region between residues 250 and 350 of human A Disintegrin-like and Metalloprotease (reprolysin type) with Thrombospondin type 1 motif, 13 using the numbering given in entry XP_088414.1 (GeneID 11093).
Specificité This antibody is specific for human ADAM TS13 protein.
Purification Immunogen affinity purified
Plasmids, Primers & others Plasmids, Primers & others ADAMTS13 products on genomics-online (e.g. as negative or positive controls)
Antigène
Autre désignation ADAMTS13 (ADAMTS13 Antibody Extrait)
Sujet Gene Symbol: ADAMTS13
ID gène 11093
NCBI Accession XP_088414
Indications d'application Western Blot 1:100-1:1000NB 100-584 has been shown to react with purified recombinant ADAM-TS13 in Western Blot. It has not been tested for reactivity with ADAM-TS13 from native sources, and the antibody is not warranted to have such activity. The suggested WB dilution is for recombinant protein.
Commentaires

The antibodies are intended for use in vitro experiments only. Our antibodies have not been tested nor are recommended for use in vivo.

Protocole Protocol specific for ADAMTS13 Antibody Nuclear Extract and Cytoplasmic Fraction Preparation:
1. Nuclear extracts (NE) and cytoplasmic fractions (S100) were prepared by Dignam's method (Dignam, Lebovitz, and Roeder, Nucleic Acids Res. 11: 1475-
. 1983).
. 100 liters of HeLa cell culture were harvested and washed 3 times with cold PBS.
. The packed-cell volume (PCV) was measured, and the cell pellet was gently resuspended with 5 PCVs of hypotonic buffer (10 mM HEPES-KOH [pH 8], 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM PMSF).
. Cells were incubated on ice for 10 minutes and then pelleted by centrifugation at 1,800xg for 10 minutes.
. Hypotonic buffer was added to 2 PCVs, and cells were resuspended and then homogenized with 15 strokes using a pestle B in a Dounce glass homogenizer until the cells were more than 90 % lysed, as determined by a light microscope.
. The lysate was centrifuged at 20,000xg for 30 minutes at 4 degrees Celcius.
. The supernatant was saved for S100 fraction, and the pellet was saved to measure the packed nuclear volume (PNV).
. 0.4 mL of extraction buffer (20 mM HEPES-KOH [pH 8], 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25 % [vol/vol] glycerol, 1 mM DTT, 0.2 mM PMSF) per mL of PNV was added.
. Cell nuclei were homogenized with 10 strokes of pestle A in the homogenizer.
. Suspension was stirred at 4 degrees Celcius for 30 minutes and centrifuged for 30 minutes at 20,000xg.
. The supernatant (nuclear extract) was aliquotted for use.
. The S100 fraction (resulting supernatant) was mixed with 0.11 volume of high-salt buffer (20 mM HEPES-KOH [pH 8], 1.2 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 % [vol/vol] glycerol, 1 mM DTT, 0.2 mM PMSF) and centrifuged at 100,000xg for 60 minutes at 4 degrees Celcius.
. This supernatant was dialyzed for 2 hours at 4 degrees Celcius.
. The sample was centrifuged for 30 minutes at 20,000xg and the supernatant (S100) was aliquotted for use.Immunoprecipitation Antibody Characterization:
. HeLa NE and S100 were diluted with 1 volume of RIPA buffer [150 mM NaCl, 1 % NP-40, 0.5 % DOC, 0.1 % SDS, 50 mM Tris [pH 8]).
. Cleared by spinning at 100,000 g for 20 minutes at 4 degrees Celcius.
. 1 mL of supernatant (~10 mg total protein) was mixed with 20 µg of primary antibody and rotated overnight at 4 degrees Celcius.
. Supernatant was mixed with 0.05 mL of protein A-sepharose beads (50 % slurry) and rotated for 2 hours at 4 degrees Celcius.
. Immunoprecipitates were washed 3 times with the 10 % RIPA in PBS.
. The washed beads were boiled with 0.04 mL of Laemmli buffer and subjected to SDS-PAGE (4-20 % Tris-glycine gel).Complex purification:
. NE and S100 were cleared by spinning at 20,000 g for 30 minutes at 4 degrees Celcius.
. 1.5 mL of supernatant (~15 mg total protein) was mixed with 20 µg of primary antibody and rotated for 4 hours at 4 degrees Celcius.
. Sample and antibody mixture were centrifuged at 15,000 g for 20 minutes at 4 degrees Celcius.
. Supernatant was mixed with 0.05 mL of protein A-sepharose beads (50 % slurry) and rotated for 1 hour at 4 degrees Celcius.
. Immunoprecipitates were washed 3 times with the NETN buffer (20 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5 % NP-40).
. The washed beads were boiled with 0.04 mL of Laemmli buffer and subjected to SDS-PAGE (4-20 % Tris-glycine gel).*If an insufficient amount of protein is purified for identification from 15 mg of extract, carry out the same procedure using 50-100 mg of extract to increase the amount of purified protein yield.
Restrictions For Research Use only
Format Liquid
Concentration 1.0 mg/mL
Buffer Tris-Citrate/Phosphate ( pH 7.0 - 8.0)
Buffer contains: 0.09 % Sodium Azide
Agent conservateur Sodium azide
Précaution d'utilisation This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Conseil sur la manipulation Do not freeze.
Stock 4 °C
Stockage commentaire Store at 4°C. Do not freeze.
Images (Fournisseur)
Western Blotting (WB) image for anti-ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 13 (ADAMTS13) (AA 250-350) antibody (ABIN152016) Detection of Recombinant Human Adam-TS13 by Western Blot. Samples: Purified recombina...
Western Blotting (WB) image for anti-ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 13 (ADAMTS13) (AA 250-350) antibody (ABIN152016) Western Blot: ADAMTS13 Antibody - Purified recombinant Adam-TS13. Antibody used at t...
Produit citée dans: Hunt, Geetha, Allen, Hershko, Fathke, Kong, Plum, Struble, Soejima, Friedman, Garfield, Balaji, Kimchi-Sarfaty: "Detection of a secreted metalloprotease within the nuclei of liver cells." dans: Molecular bioSystems, Vol. 7, Issue 6, pp. 2012-8, 2011 (PubMed).

Chung, Popova, Jorgensen, Dong, Chandhoke, Bailey, Popov: "Degradation of circulating von Willebrand factor and its regulator ADAMTS13 implicates secreted Bacillus anthracis metalloproteases in anthrax consumptive coagulopathy." dans: The Journal of biological chemistry, Vol. 283, Issue 15, pp. 9531-42, 2008 (PubMed).

Tao, Wang, Choi, Bernardo, Nishio, Sadler, López, Dong: "Cleavage of ultralarge multimers of von Willebrand factor by C-terminal-truncated mutants of ADAMTS-13 under flow." dans: Blood, Vol. 106, Issue 1, pp. 141-3, 2005 (PubMed).

Dong: "Cleavage of ultra-large von Willebrand factor by ADAMTS-13 under flow conditions." dans: Journal of thrombosis and haemostasis : JTH, Vol. 3, Issue 8, pp. 1710-6, 2005 (PubMed).

Tao, Peng, Nolasco, Cal, Lopez-Otin, Li, Moake, López, Dong: "Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions." dans: Blood, Vol. 106, Issue 13, pp. 4139-45, 2005 (PubMed).

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