NCOA4 anticorps (N-Term)

N° du produit ABIN2780237

Détail du produit anti-NCOA4 anticorps

Antigène: NCOA4 (Nuclear Receptor Coactivator 4 (NCOA4))

Épitope: N-Term

Reactivité: Humain, Souris, Rat, Boeuf (Vache), Chien, Cheval, Cobaye, Lapin

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp NCOA4 est non-conjugé

Application: Western Blotting (WB)

Séquence: CLIHQLEYTQ NKDLANQVSV CLERLGSLAL KPEDSTVLLF EADTSALRQT

Homologie: Cow: 92%, Dog: 93%, Guinea Pig: 100%, Horse: 93%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%

Attributs du produit: This is a rabbit polyclonal antibody against Ncoa4. It was validated on Western Blot.

Purification: Affinity Purified

Immunogène: The immunogen is a synthetic peptide corresponding to a region of Mouse

Information d'application

Indications d'application: Optimal working dilutions should be determined experimentally by the investigator.

Commentaires:

Antigen size: 625 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100057 (Western Blotting)

by: University of Michigan Medical School, Molecular & Integrative Physiology, Shah lab

No.: #100057

Date: 12.09.2016

Antigène: Ncoa4

Numéro du lot: QC11327

Application validée: Western Blotting

Contrôle positif: HIF overexpression mice, WT mice

Contrôle négative: NCOA4-/- mice

Conclusion: Passed, based on the fact that the antibody picked up the positive control (unpublished data suggests that NCOA4 is highly expressed in an intestinal HIF overexpression model).

Validation Images

Mouse duodenum whole cell lysates from intestinal HIF overexpression (HIF OE) mice, NCOA4-/- mice, and wt mice fed a normal or low iron diet were separated by SDS-PAGE. NCOA4 was visualized using ABIN2780237. A ferritin (Ftn) Western blot was done to confirm the efficacy of iron diet regimens in mice. GAPDH served as loading control.

Protocole

Anticorps primaire: ABIN2780237

Anticorps secondaire: Anti-rabbit HRP-linked secondary antibody (Cell Signaling, 7074S, lot 26)

Full Protocol:

• Wild type (WT) or NCOA4 -/- mice were fed normal or iron-deficient diet for 7 days, followed by euthanization and scraping of duodenal epithelium (DE).
• DE cells were lysed in RIPA buffer and whole cell lysate (WCL) prepared.
• 50µg total protein from each sample were used for Western blot analysis.
• Samples were denatured in 5x gel-loading buffer containing beta-marcaptoethanol and separated on a 10% SDS-PAGE gel (Laemmli 1970).
• Transfer onto Nitrocellulose membrane (Biorad, 1620212, lot 10484060) was done on Trans-Blot SD apparatus (Bio-Rad) following the manufacturer’s protocol.
• Membranes were blocked with TBS-tween (TBST) solution containing 5% milk for 1h.
• Incubation with primary antibody overnight at 4°C:
• • NCOA4 antibody ABIN2780237 (lot QC11327) diluted 1:1000 in TBST containing 0.1% BSA.
  • FTH1 antibody (Cell signaling, 3998S; lot 1) diluted in TBST containing 0.1% BSA.
  • GAPDH antibody (Santa Cruz, SC-25778, lot D30115) diluted in TBST containing 0.1% BSA.
• Washing with TBST for 15min at RT.
• Incubation with anti-rabbit HRP-linked secondary antibody (Cell Signaling, 7074S, lot 26) diluted 1:2000 in TBST a shaker for 1h at RT.
• Three rinses, followed by four 15min washes, twice TBST and twice with TBS.
• Chemiluminescence detection was done using ECL Select Western blotting detection reagent (Amersham, RPN2235, lot 9723881).
• Blot was developed on a Biorad imaging system.

Notes:

• The antibody did not consistently pick up the WT mice on normal and low iron diet.
• Also, there is still a faint band in the NCOA4 -/- lane. We are confident that it is a true KO mouse, as there is high ferritin expression even after 7 days of low iron diet (compared to ferritin expression in WT low iron mice as well as that of the HIF over expression mouse, which are known to have high intestinal ftn content).

Stockage

Format: Liquid

Concentration: Lot specific

Buffer: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

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: Avoid repeated freeze-thaw cycles.

Stock: -20 °C

Stockage commentaire: For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.

Détails sur NCOA4

Antigène: NCOA4 (Nuclear Receptor Coactivator 4 (NCOA4))

Autre désignation: Ncoa4 (NCOA4 Produits)

Sujet: The function of Ncoa4 remains unknown.
Alias Symbols: 1110034E15Rik, 4432406M01Rik, AI227008, ARA70, MGC103382, Rfg
Protein Interaction Partner: Ndn,
Protein Size: 625

Poids moléculaire: 70 kDa

ID gène: 27057

NCBI Accession: NM_001033988, NP_001029160

Pathways: Intracellular Steroid Hormone Receptor Signaling Pathway