SYNGAP1 anticorps (Internal Region)

N° du produit ABIN955030

Détail du produit anti-SYNGAP1 anticorps

Antigène: SYNGAP1 (Synaptic Ras GTPase Activating Protein 1 (SYNGAP1))

Épitope: AA 1169-1183, Internal Region

Reactivité: Humain, Souris, Rat, Boeuf (Vache), Chien, Porc

Hôte: Chèvre

Clonalité: Polyclonal

Conjugué: Cet anticorp SYNGAP1 est non-conjugé

Application: Western Blotting (WB), Enzyme Immunoassay (EIA)

Specificité: This antibody reacts to SYNGAP1.

Réactivité croisée (Details): Species reactivity (expected):Human, Rat, Canine, Porcine and Bovine.
Species reactivity (tested):Mouse.

Purification: Affinity Purified

Immunogène: Peptide with sequence C-ESAHIEREEYKLKEY, from the internal region (near C Terminus) of the protein sequence according to NP_006763.2.

Information d'application

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

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100923 (Western Blotting)

by: Molekulare Neurobiologie, Max-Planck-Institut für Experimentelle Medizin

No.: #100923

Date: 08.04.2017

Antigène: SYNGAP1

Numéro du lot: P1

Application validée: Western Blotting

Contrôle positif: SYNGAP1 transfected HEK293TF cells, anti-SYNGAP1 antibody PA1-046

Contrôle négative: untransfected HEK293TF cells

Conclusion: Passed. ABIN955030 specifically detects SynGAP in transiently transfected HEK293TF cells expressing murine SynGAP.

Validation Images

HEK293TF cell transfected with a SynGAP expressing (+) or empty pcDNA3 plasmid (-) were lysed and extracted proteins separated in duplicates on a poly-acrylamine gel and transferred to nitrocellulose membrane. Separated proteins were incubated either with the anti-SYNGAP1 antibody ABIN955030 (A) or a different anti-SynGAP antibody (B) at the indicated dilutions. Protein transfer to the nitrocellulose membranes was verified by PonceauS staining prior to the immunoblot (C, D).

Protocole

Anticorps primaire: ABIN955030

Anticorps secondaire: mouse anti-goat HRP-conjugate (Sigma)

Full Protocol:

• HEK293TF cells are grown in in 10 cm cell culture dishes in DMEM (1x) + Glutamax TM-I (Gibco, 31966-021, lot 1838117) supplemented with 10% fetal bovine serum Gibco, 105000064, lot 08F7660K) and 0.5% Penicillin/Streptomycin (Gibco, 15140122, lot 1864845)at 37°C in 5% CO₂.
• Transfect cells at 50% confluence with plasmid encoding full-length murine SynGAP or empty pcDNA3(+) vector as negative control using Lipofectamine 2000 (Invitrogen, 1375188/5000, lot 835373) following the manufacturer’s instructions. Dilute the transfection reagent plasmids in Opti-MEM Reduced Serum Media (Gibco) and applied following manufacturer's instructions.
• Change growth media after 5h.
• Grow cells for 48h.
• Wash cells with PBS.
• Trypsinize and transfer cells to 2ml microcentrifuge tubes.
• Wash cell pellet 1x with PBS.
• Sonicate cells with a Bandelin Sonopuls Gm sonicator in ice cold lysis buffer (20mM TrisHCl, 150mM NaCl pH8.0, 1% NP-40) containing protease Inhibitors (1μg/mL aprotinin, 0.5μg/mL leupeptine, 17.4μg/mL PMSF).
• Centrifuge samples in an ultracentrifuge (Beckmann) in TLA-100.3 rotor at 50000 rpm (135000xg) for 1h at 4°C to clear samples from cellular debris.
• Transfer the supernatants to 2ml microcentrifuge tubes add an equal volume of 2x Laemmli buffer (100mM Tris, 4% SDS, 0.2% Bromphenol Blue [Pierce], 20% Glycerol, 200mM DTT, pH6.8) added and denature samples for 5min at 95°C.
• Separate proteins on freshly cast 10% polyacrylamide gels for 2h at 80V.
• Transfer proteins onto nitrocellulose membranes (GE Healthcare, 10600001, A100773742) in a tank blot for 16h at 45mA.
• Before immunoblotting, stain membranes with PonceauS (0.5% PonceauS, 1% acetic acid, Sigma-Aldrich) to verify transfer.
• Wash membranes 1x with PBST (0.1% Tween20 in PBS).
• Block membranes with PBST containing 5% milk powder for 1h at RT.
• Incubation with primary antibodies
• • goat anti-SYNGAP1 antibody (antibodies-online, ABIN955030, lot P1) diluted 1:1000 or
  • rabbit anti SynGAP antibody (Thermo Scientific, PA1-046, lot RE235674) diluted 1:5000
• in PBST containing 5% milk powder for 2h at RT.
• Wash membranes 3x 5min with PBST (+5% milk powder).
• Incubation with secondary antibody
• • mouse anti-goat HRP-conjugate (Sigma) or
  • goat anti-Rabbit IgG (H+L)-HRP Conjugate (Bio-Rad, 1721019)
• diluted 1:10000 in PBST containing 5% milk powder for 1h at RT.
• Wash membranes 3x 5min with PBST.
• Reveal proteins using the ECL Western blotting detection system (GE Healthcare, PPN2106V1/V2, lot 9770467) and GE Healthcare Hyperfilm.

Notes: ABIN955030 reveals a predominant protein band of the expected molecular weight of murine SynGAP in lysates HEK293TF transiently expressing the protein.

Stockage

Concentration: 0,5 mg/mL

Buffer: Tris saline, pH 7.3 containing 0.02 % sodium azide as preservative and 0.5 % bovine serum albumin as stabilizer

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 freezing and thawing.

Stock: 4 °C/-20 °C

Stockage commentaire: Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.

Détails sur SYNGAP1

Antigène: SYNGAP1 (Synaptic Ras GTPase Activating Protein 1 (SYNGAP1))

Autre désignation: SYNGAP1 (SYNGAP1 Produits)

Synonymes: anticorps SYNGAP1, anticorps Syngap, anticorps MRD5, anticorps RASA1, anticorps RASA5, anticorps SYNGAP, anticorps Gm1963, anticorps synaptic Ras GTPase activating protein 1, anticorps zinc finger and BTB domain containing 9, anticorps synaptic Ras GTPase activating protein 1 homolog (rat), anticorps SYNGAP1, anticorps ZBTB9, anticorps syngap1, anticorps Syngap1

Sujet: Synonyms: KIAA1938, Neuronal RasGAP, RASA1, RASA5, Ras GTPase-activating protein SynGAP, SYNGAP, Synaptic Ras-GAP 1

ID gène: 8831

NCBI Accession: NP_006763

Pathways: Regulation of long-term Neuronal Synaptic Plasticity