On DIV6, 12, 16, or 20, cells were collected in 125µL PBS and 125µL Laemmli sample buffer (BioRad). 15µL of cell lysate was run on an SDS/PAGE gel and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo transfer system (BioRad). Primary antibodies were used at the following concentrations: GAPDH (14C10; Cell Signaling) 1:50,000; synaptobrevin2 (104 211; Synaptic Systems) 1:5,000; synaptotagmin1 (105 011; Synaptic Systems) 1:5,000; synapsin1 (106 103; Synaptic Systems) 1:2,000; Vti1a (611220; BD Biosciences) 1:500. After incubation in primary antibodies at 4°C overnight, membranes were incubated in secondary antibodies at the following concentrations: GAPDH 1:10,000 (anti-rabbit); synaptobrevin2 1:5,000 (anti-mouse); synaptotagmin1 1:5,000 (anti-mouse); synapsin1 1:5,000 (anti-rabbit); Vti1a 1:5,000 (anit-mouse). Protein bands were detected using ECL and exposure to film. Quantification was performed using ImageJ to detect protein band intensities. Values were normalized to GAPDH before comparison.
Synaptotagmin 1
Manufactured by Synaptic Systems
Sourced in Germany
Synaptotagmin 1 is a membrane-bound protein that plays a central role in the regulation of neurotransmitter release from synaptic vesicles. It acts as a calcium sensor, triggering the fusion of synaptic vesicles with the presynaptic membrane upon an increase in intracellular calcium concentration.
Automatically generated - may contain errors
Lab products found in correlation
Snap25, by Synaptic Systems (2 mentions)
Vti1a, by BD (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Synaptobrevin 2, by Synaptic Systems (1 mentions)
Synaptophysin, by Merck Group (1 mentions)
Secondary antibody conjugated to horseradish peroxidase, by GE Healthcare (1 mentions)
Actin, by Merck Group (1 mentions)
Infrared dye conjugated secondary antibody, by LI COR (1 mentions)
Glur2, by Merck Group (1 mentions)
Glur1, by Merck Group (1 mentions)
Syntaxin 1, by Merck Group (1 mentions)
Vamp2, by Synaptic Systems (1 mentions)
Na k pump, by Abcam (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Lamp1, by Developmental Studies Hybridoma Bank (1 mentions)
Ultimate 3000 chromatography system, by Thermo Fisher Scientific (1 mentions)
Synaptophysin, by Synaptic Systems (1 mentions)
Syntaxin 1, by Synaptic Systems (1 mentions)
Homer1, by Synaptic Systems (1 mentions)
5 protocols using synaptotagmin 1
1
Quantitative Immunoblotting of Synaptic Proteins
2
Comprehensive Synaptic Protein Expression
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We used the following antibodies at the titer indicated: VGLUT1 (1:2000), VGLUT2 (1:1000), VGAT (1:2000), SV2 (Developmental Studies Hybridoma Bank, 1:1000), synaptophysin (Sigma, 1:2000), synaptotagmin 1 (Synaptic Systems, 1:1000), VAMP2 (Synaptic Systems, 1:1000), syntaxin 1 (Sigma, 1:1000), SNAP25 (Synaptic Systems, 1:1000), munc18 (BD Biosciences, 1:500), GluR1 (Millipore 1:1000), GluR2 (Millipore, 1:500), NR1 (BD Biosciences, 1:1000), PSD-95 (Neuromab, 1:1000), TfR (Invitrogen, 1:500), rab3 (Synaptic Systems, 1:500), rab5 (Synaptic Systems, 1:500), rab7 (Sigma, 1:500), LAMP1 (Developmental Studies Hybridoma Bank, 1:500), V-ATPase H subunit (Santa Cruz, 1:100), Na+/K+ pump (Abcam, 1:1000), α-ATP synthase (Abcam, 1:1000), GFAP (Zymed, 1:250), CPE-c term (P. Loh, at 1:500), actin (Millipore, 1:1000), FLAG (Sigma, 1:2000), voltage-gated calcium channel subunit α1A (Synaptic Systems, 1:500), voltage-gated calcium channel subunit α2δ-1 (Sigma, 1:1000), EAAT1 (Synaptic Systems, 1:1000) and PrPc (S. Prusiner D13, 1:1000). Infrared dye-conjugated secondary antibodies (LI-COR) were used for western blotting at 1:50,000 with the exception of the calcium channel subunit α1A and PrPc, which required chemiluminescence detection using secondary antibody conjugated to horseradish peroxidase at 1:5000 (GE Healthcare).
3
Parallel Quantification of Synaptic Proteins
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High-resolution parallel reaction monitoring was performed on a Q Exactive quadropole-orbitrap mass spectrometer coupled to an Ultimate 3000 chromatography system (Thermo Fisher Scientific) for the parallel quantification of SNAP-25 (amino acids 32-40 = SNAP-25aa40 and Ac-2-16 = SNAP-25tot) and synaptotagmin-1 (amino acids 215-228) in CSF. Antibodies included mouse monoclonal antibody SP12 recognising SNAP-25 and mouse monoclonal antibody SM181 recognising an epitope containing the N-terminally acetylated first 11 amino acids of SNAP-25 (as previously described by Brinkmalm et al. [13 (link)]), and monoclonal antibody clone 41.1 recognising the cytoplasmic portion of synaptotagmin-1 from Synaptic Systems (detailed methods previously described by Öhrfelt et al. [14 (link)] and Fernström et al. [18 (link)]).
4
Immunoblotting of Synaptic Proteins
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Antibodies were directed against APLP1 (CT-11, rabbit polyclonal, 1:2000, Calbiochem—Merck Millipore, Darmstadt, Germany) APP (Y188, rabbit monoclonal, 1:1000, Abcam, Cambridge, UK), Munc18 (rabbit polyclonal, 1:1000 Synaptic Systems, Göttingen, Germany), NCAM (rabbit polyclonal, 1:1000, Abcam, Cambridge, UK), SNAP25 (mouse monoclonal 1:1000 Synaptic Systems, Göttingen, Germany), synaptophysin (G63, rabbit polyclonal, 1:1000, kindly donated by Dr. R. Jahn, Göttingen, Germany), synaptotagmin-1 (rabbit polyclonal, 1:1000, Synaptic System, Göttingen, Germany), syntaxin-1 (mouse monoclonal, 1:1000 Synaptic Systems, Göttingen, Germany), SV2 (the clone CKK 10H4 producing the monoclonal anti-SV2 antibody, kindly donated by Dr. Regis B. Kelly, San Francisco, CA, USA; was cultured in-house), SV2A (rabbit polyclonal, 1:1000 kindly donated by Dr. S. Bajjalieh, Seattle, USA), and VAMP2 (cl. 69.1, mouse monoclonal, 1:1000 Synaptic Systems, Göttingen, Germany).
Dynabeads M-280 conjugated with monoclonal sheep anti-mouse IgGs (cat. No. 112.02D) were purchased from Invitrogen, Darmstadt, Germany.
Dynabeads M-280 conjugated with monoclonal sheep anti-mouse IgGs (cat. No. 112.02D) were purchased from Invitrogen, Darmstadt, Germany.
5
Molecular Markers of Synaptic Signaling
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PI3K-C2β (BD Biosciences, #611342, 1:1000), phospho-p70 S6 kinase (Thr389; Cell Signaling, #9205, 1:500), p70 S6 kinase (Cell Signaling, #9202, 1:1000), S6 ribosomal protein (Cell Signaling, #2217, 1:1000), phospho-S6 ribosomal protein (Ser235/236; Cell Signaling, #2211, 1/1000), phospho-Akt (Ser473) antibody (Cell Signaling, #9271, 1:1000), Akt (pan; Cell Signaling, #4691, 1:1000), GAPDH (Cell Signaling, #2118, 1/2500), PSD95 (Synaptic Systems, #124 011, 1:1000), vGAT (Synaptic Systems, #131011, 1:1000), vGLUT (Synaptic Systems, #135302, 1:1000), synaptotagmin 1 (1Synaptic Systems, #105 011, 1:5000), mTOR (Cell Signaling, #7C10, 1:1000), GluA1 (Millipore, # MAB2263, 1:1000), Homer1 (Synaptic Systems, #160003, 1:1000), Rab7 (Cell Signaling, #9367, 1:1000), p62 (Cell Signaling, #5114, 1:1000), LC3 (Abcam, #ab48394, 1:1000), GFP (Abcam, #ab290, 1:1000), Vinculin (Abcam, #ab129002, 1:5000).
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