RAB11A anticorps (C-Term)

N° du produit ABIN2346690

Cet anticorps anti-RAB11A est un anticorps Lapin Polyclonal détectant RAB11A dans WB, ELISA, IHC, IF et IP. Adapté pour Humain. Validé indépendamment pour une utilisation dans Western Blotting.

Aperçu rapide pour RAB11A anticorps (C-Term) (ABIN2346690)

Antigène: RAB11A (RAB11A, Member RAS Oncogene Family (RAB11A))

Reactivité: Humain

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp RAB11A est non-conjugé

Application: Western Blotting (WB), ELISA, Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP)

Détail du produit anti-RAB11A anticorps

Épitope: C-Term

Réactivité croisée: Chien, Humain, Lapin, Rat (Rattus)

Réactivité croisée (Details): Calculated cross reactivity: Ca Hu Rb Rt

Attributs du produit: Rab11A (RAB11A Member RAS Oncogene Family, Rab-11A, MGC1490, RAB11, Rab-11, Ras-related Protein Rab-11A, Ras-related protein Rab11A, YL8)

Purification: Purified by immunoaffinity chromatography.

Immunogène: Synthetic peptide corresponding to the C-terminal hypervariable region of the human Rab11a sequence. Sequence Homology: Rab11b protein-10/17.

Isotype: IgG

Information d'application

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

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101022 (Western Blotting)

by: Epidermis Differentiation and Rheumatoid Autoimmunity (INSERM U1056 UDEAR), University of Toulouse

No.: #101022

Date: 27.07.2017

Antigène: RAB11A

Numéro du lot: L16102562

Application validée: Western Blotting

Contrôle positif:

Cell lysates from total human epidermis

Cell lysates from reconstructed human epidermis transfected with control shRNA

Contrôle négative:

Cell lysates from reconstructed human epidermis transfected with RAB11A shRNA

Conclusion: Passed. ABIN2346690 specifically recognizes Rab11a in human epidermal samples.

Validation Images

Western blot analysis of reconstructed human epidermis cell lysates with ABIN2346690 reveals a band of the expected size for Rab11a in the lysates of the cells transfected with a scrambled control shRNA (left lane) which is absent in lysates from cells that were transfected with RAB11A shRNA. Total human epidermis lysates (right lane) served as control. The immunoblot with ABIN2346690 reveals the expected protein band.

Protocole

Anticorps primaire: ABIN2346690

Anticorps secondaire: goat anti-rabbit Ig HRP-conjugated antibody (Southern Biotech, 4030-05, lot J1810)

Full Protocol:

• Obtain total human epidermis from abdominal dermolipectomy and treat with dispase (Sigma-Aldrich, D4693) to dissociate it from the dermis.
• For production of reconstructed human epidermis, obtain isolated keratinocytes from epidermal fragments. Grow cells in Epilife (Invitrogen Life Technologies, MEPICF500, lot 1838688) supplemented with penicillin and streptomycin (Sigma-Aldrich, P4333, lot 1868847), HKGS (Human Keratinocyte Growth Supplement, ThermoFisher Scientific, S0015, lot 1848268), 1.5mM calcium (Sigma-Aldrich 25011), KGF (Keratinocyte Growth Factor, Sigma-Aldrich, K1767, lot MKBX7825V) and vitamin C (Sigma-Aldrich, 49752) on polycarbonate culture inserts (Merck Millipore, PIHP01250) at 37°C and 5% CO₂, according to Reynier et al. (2016).
• For silencing with interfering shRNA, transduce keratinocytes with MISSION pLKO.1 puro vector-based lentiviral particles containing a puromycin resistance gene and an shRNA insert under the human U6 promoter (Sigma-Aldrich, TRCN0000303291 for Rab11A and SHC002 for the negative control) following the manufacturer´s instructions, before cell seeding for production of reconstructed human epidermis.
• Lyse normal or reconstructed epidermis in 300µL/0.6cm² 1x Laemmli buffer (Tris-HCl 63mM pH6.8, SDS 2%, Glycerol 10%, Bromophenol blue 0.0005%, DTT 1mM, all Sigma-Aldrich) and heat at 95°C for 5min.
• Separate 5µL of total protein extracts in 1x Laemmli buffer on denaturing 10% polyacrylamide SDS gels in a Mini-Protean Tetra Cell electrophoresis cell system (Bio-Rad , 1658004) at 100V for 90min.
• Transfer proteins onto Hybond ECL membrane (GE Healthcare, RPN3032D) with a Mini TransBlot Western blotting system (Bio-Rad, 1658029) at 100V for 90 min.
• Block membrane with 1x PBS containing Tween 20 (Sigma-Aldrich) at 0.05% (PBST) and 5% low fat milk at 3% at RT for 1h.
• Incubate the membrane with primary rabbit anti-human RAB11A, Member RAS Oncogene Family (RAB11A) (C-Term) antibody (antibodies-online, ABIN2346690, lot L16102562) diluted 1:250 in blocking buffer ON at 4°C.
• Wash the membrane 3x for 15min with 1x PBST.
• Incubate the membrane with secondary goat anti-rabbit Ig HRP-conjugated antibody (Southern Biotech, 4030-05, lot J1810) diluted 1:10000 in blocking buffer for 90min at RT.
• Wash the membrane 3x for 15min with 1x PBST.
• Reveal protein bands using ECL prime Detection Reagent (GE Healthcare, RPN2238, lot 9838391) on a GBox apparatus (Syngene).

Stockage

Format: Liquid

Buffer: Supplied as a liquid in PBS, pH 7.4, 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.

Stock: -20 °C

Stockage commentaire: -20°C

Détails sur RAB11A

Antigène: RAB11A (RAB11A, Member RAS Oncogene Family (RAB11A))

Autre désignation: Rab11A

Pathways: Regulation of Cell Size, Thromboxane A2 Receptor Signaling, Regulation of long-term Neuronal Synaptic Plasticity