SYNGAP1 antibody (Internal Region)

Catalog No. ABIN955030

Product Details anti-SYNGAP1 Antibody

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

Binding Specificity: AA 1169-1183, Internal Region

Reactivity: Human, Mouse, Rat, Cow, Dog, Pig

Host: Goat

Clonality: Polyclonal

Conjugate: This SYNGAP1 antibody is un-conjugated

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

Specificity: This antibody reacts to SYNGAP1.

Cross-Reactivity (Details): Species reactivity (expected):Human, Rat, Canine, Porcine and Bovine.
Species reactivity (tested):Mouse.

Purification: Affinity Purified

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

Application Details

Application Notes: 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: 04/08/2017

Antigen: SYNGAP1

Lot Number: P1

Method validated: Western Blotting

Positive Control: SYNGAP1 transfected HEK293TF cells, anti-SYNGAP1 antibody PA1-046

Negative Control: untransfected HEK293TF cells

Notes: 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).

Full Methods

Primary Antibody: ABIN955030

Secondary Antibody: 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.

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

Handling

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

Preservative: Sodium azide

Precaution of Use: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Avoid repeated freezing and thawing.

Storage: 4 °C/-20 °C

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

Target Details for SYNGAP1

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

Alternative Name: SYNGAP1 (SYNGAP1 Products)

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

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

Gene ID: 8831

NCBI Accession: NP_006763

Pathways: Regulation of long-term Neuronal Synaptic Plasticity