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).
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.
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
SYNGAP1
(Synaptic Ras GTPase Activating Protein 1 (SYNGAP1))