Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 (CYP4B1) (N-Term) anticorps

Détails pour le produit réf. ABIN2777006, Fournisseur: Connectez-vous pour afficher
Antigène
  • cyp4b1
  • CYPIVB1
  • P-450HP
  • cytochrome P450, family 4, subfamily B, polypeptide 1
  • CYP4B1-like isozyme short form
  • cytochrome P450, family 4, subfamily b, polypeptide 1
  • CYP4B1
  • cyp4b1.2
  • cyp4b1
  • Cyp4b1
Épitope
N-Term
8
7
4
4
2
2
1
1
1
1
Reactivité
Boeuf (Vache), Chien, Chévre, Cobaye, Cheval, Humain, Souris, Lapin, Rat (Rattus)
39
9
8
2
2
2
2
2
1
1
1
Hôte
Lapin
35
4
Clonalité
Polyclonal
Application
Western Blotting (WB)
35
13
11
5
4
1
1
1
Options
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Supplier Product No.
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Images

'Independent Validation' signe
Antigène CYP4B1
Numéro du lot QC12731-90422
Application validée Immunofluorescence
Contrôle positif WT lung small airway sections
Contrôle négative WT lung treated with naphthalene
Conclusion The antibody ABIN2777006 specifically stains CYP4B1 in fixed murine respiratory tissue.
Anticorps primaire ABIN2777006
Anticorps secondaire donkey anti-rabbit AF594 conjugated antibody (Invitrogen)
Protocole
  • Naphthalene treatment: i.p. application of 200mg per kg body weight (KG) naphthalene dissolved in oil.
  • Fix mouse lung tissue in 4% PFA /PBS ON at RT on a shaker.
  • Wash tissue 2-3x 60min with tap water/PBS.
  • Place tissue in
    • 70% EtOH 2x 15min or more
    • 80% EtOH 2x 30min
    • 90% EtOH 3x 30min
    • 100% ETOH 2x 90min
    • isopropanol ON
    • 25% Xylol/75% isopropanol 60min
    • 50% Xylol/50% isopropanol 60min
    • 75%Xylol/25% isopropanol 60min
    • xylol until clear (looks like jelly) ON
    • paraffin at 60°C for 3d changing every day
  • Embed tissue in paraffin and cool down to 4°C for a few minutes.
  • Cut blocks into 3µm sections using a semi-automated rotary microtome from Leica (RM2245).
  • Deparaffinize sections in:
    • xylol 2h
    • 50% xylol/50% isopropanol 10min
    • 100% isopropanol 10min
    • 100% EtOH 5min
    • 90% EtOH 5min
    • 70% EtOH 5min
  • Wash sections briefly in distilled water.
  • Boil sections in 10mM citric acid buffer pH6.0 in a microwave for 20min.
  • Rinse sections in distilled water.
  • Wash sections 2x 5min in PBS.
  • Block sections in blocking solution (PBS, 0.05% Tween-20, 10% FCS) for 45min-60min at RT.
  • Wash sections 2x 5min in PBS.
  • Incubate sections with 70µl for each section primary
    • rabbit anti-cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1) (N-Term) antibody (antibodies-online, ABIN2777006, lot QC12731-90422) diluted 1:50
    • mouse anti-alpha-tubulin antibody (Sigma, T 6793) diluted 1:1000
  • in blocking solution for 16h at 4°C in a humidified chamber.
  • A no primary antibody negative control was incubated similarly in parallel.
  • Rinse sections 3x 5min with PBS.
  • Incubate sections with secondary
    • donkey anti-rabbit AF594 conjugated antibody (Invitrogen)
    • donkey anti-mouse AF488 conjugated antibody (Invitrogen)
  • diluted 1:500 in blocking solution for 1h at room temperature.
  • Wash sections 1x 5min with PBS containing 1:2000 diluted 1mg/ml DAPI stock solution.
  • Rinse sections 2x with PBS.
  • Mount slides with mounting medium (Dako).
  • Image acquisition with the same exposure and intensity for all IFs.
Notes
  • Staining of CYP4B1 with ABIN2777006 is visible in mouse WT lung small airway sections but not in negative control sections of naphthalene treated lungs in which club cells must have been depleted by apoptosis.

  • Club cells in the respiratory system are stained by ABIN2777006 in the smaller airways rather than big airways which is consistent with our literature knowledge. The Cyp450 antibody stains the non-ciliated cells, fitting the hypothesis perfectly. Pear-shaped stained cells reaching into the lumen of bronchiole also supports this finding.

Validation Images
Immunofluorescence validation image for anti-Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 (CYP4B1) (N-Term) antibody (ABIN2777006) Staining of mouse WT bronchioles with CYP4B1 antibody ABIN2777006 (red), DAPI (blue),...
'Independent Validation' signe
Antigène CYP4B1
Numéro du lot QC12731-90422
Application validée Western Blotting
Contrôle positif Lung lysates from untreated mice
Contrôle négative Lung lysates from naphthalene treated mice
Conclusion ABIN2777006 successfully reveals Cyp4b1 in murine respiratory tissue lysates.
Anticorps primaire ABIN2777006
Anticorps secondaire donkey anti-rabbit IgG (H+L) peroxidase-conjugated F(ab')2 fragment (Jackson ImmunoResearch)
Protocole
  • Naphthalene treatment: i.p. application of 200mg per kg body weight (KG) naphthalene dissolved in oil.
  • Denature 100µg total protein for 5min at 95°C in Laemmli SDS sample buffer and subsequently separate them on a denaturing gel SDS-PAGE gel in for 90min at 100V.
  • Transfer proteins onto 0.2µm nitrocellulose membrane in transfer buffer (15% MeOH, Tris 25mM, 86mM, SDS 70mM).
  • Block the membrane with blocking solution (PBS, 5% milk).
  • Incubation with primary
    • rabbit anti-cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1) (N-Term) antibody (antibodies-online, ABIN2777006, lot QC12731-90422) diluted 1:300 or
    • Hsc70 loading control antibody (supplier, product no, lot no) diluted
  • in blocking solution ON at 4°C.
  • Wash membrane 3x with blocking solution.
  • Incubate with secondary donkey anti-rabbit IgG (H+L) peroxidase-conjugated F(ab')2 fragment (Jackson ImmunoResearch) diluted1:10000 in blocking solution for 1h at RT.
  • Wash membrane 3x with PBS, 1x with blocking solution, 2x with PBS.
  • Develop membrane with SuperSignal West femto substrate (Thermo Fisher Scientific, 34095) according to manufacturer’s recommendations, exposure time 25min.
  • Repeat the incubation steps with both antibodies, washes, and revealing the protein bands.
Notes
  • Staining of Cyp4b1 in murine lung lysates by ABIN2777006 was very weak with considerable background after one incubation of the membranes with the antibody under the described conditions but did show a band of the expected MW at 59kDa. After a second round of incubation with both primary and secondary antibody this band was much stronger compared to the background.

  • The 59kDa band is diminished in lysates taken two days after naphthalene treatment normalized to Hsc70. Its intensity increases again at day three.

  • ABIN2777006 does also reveal a larger band than 59kDa which might represent ubiquitinylated or SUMOylated Cyp4b1. This would be consistent with this band being strongest 2d after the naphthalene treatment.

Validation Images
Western Blotting validation image for anti-Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 (CYP4B1) (N-Term) antibody (ABIN2777006) Detection of Cyp4b1 by ABIN2777006 in untreated mouse respiratory tissue lysates from...
Immunogène The immunogen is a synthetic peptide directed towards the N terminal region of human CYP4B1
Blocking Peptide Blocking peptide for this product available: ABIN975554
Séquence SWAHQFPYAH PLWFGQFIGF LNIYEPDYAK AVYSRGDPKA PDVYDFFLQW
Homologie Cow: 100%, Dog: 100%, Goat: 93%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%
Attributs du produit This is a rabbit polyclonal antibody against CYP4B1. It was validated on Western Blot using a cell lysate as a positive control.
Purification Affinity Purified
Autre désignation CYP4B1 (CYP4B1 Antibody Extrait)
Sujet CYP4B1 is a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum. In rodents, the homologous protein has been shown to metabolize certain carcinogens, however, the specific function of the human protein has not been determined.This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum. In rodents, the homologous protein has been shown to metabolize certain carcinogens, however, the specific function of the human protein has not been determined. Two transcript variants encoding slightly different isoforms have been found for this gene.
Alias Symbols: CYPIVB1, P-450HP
Protein Size: 511
Poids moléculaire 59 kDa
ID gène 1580
NCBI Accession NM_000779, NP_000770
UniProt P13584
Domaine de recherche Signaling, Metabolism
Indications d'application Optimal working dilutions should be determined experimentally by the investigator.
Commentaires

Antigen size: 511 AA

Restrictions For Research Use only
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.
Supplier Images
Western Blotting (WB) image for anti-Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 (CYP4B1) (N-Term) anticorps (ABIN2777006) anti-Cytochrome P450, Family 4, Subfamily B, Polypeptide 1 (CYP4B1) (N-Term) antibody
Background publications Hiratsuka, Nozawa, Konno, Saito, Konno, Mizugaki: "Human CYP4B1 gene in the japanese population analyzed by denaturing HPLC." dans: Drug metabolism and pharmacokinetics, Vol. 19, Issue 2, pp. 114-9, 2004 (PubMed).