Anti-GLUT1 (#652) and anti-GLUT4 (#654) were from AbCam (Cambridge, United Kingdom). Anti-GAPDH (#4300) was from Ambion (Foster city, California, USA). Anti-VAMP1 (#104002), anti-VAMP2 (#104202), anti-VAMP3 (#43080), anti-VAMP4 (#136002), anti-VAMP5 (#176003), anti-VAMP7 (#232003), anti-VAMP8 (#104302), anti-SNAP23 (#111202), anti-SNAP29 (#111303), anti-SNAP47 (#111403), anti-syntaxin2 (#110022), anti-syntaxin3 (#110032), anti-syntaxin4 (#110042), anti-syntaxin5 (#110053), anti-syntaxin7 (#110072), anti-syntaxin8 (#110083), and anti-syntaxin16 (#110162) were from Synaptic Systems (Goettingen, Germany). Anti-syntaxin6 (#610635) was from BD Biosciences (Franklin lakes, New Jersey, USA). Fluorescent secondary antibodies were from LI-COR Biosciences (Lincoln, Nebraska, USA).
Anti vamp2
Manufactured by Synaptic Systems
Sourced in Germany
Anti-VAMP2 is a primary antibody that specifically targets the VAMP2 (Vesicle-Associated Membrane Protein 2) protein. VAMP2 is a key component of the SNARE complex involved in the fusion of synaptic vesicles with the presynaptic membrane during neurotransmitter release. The Anti-VAMP2 antibody can be used to detect and study the localization and expression of VAMP2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti bassoon, by Abcam (2 mentions)
Fluorescent secondary antibody, by LI COR (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
Anti glut4, by Abcam (1 mentions)
Anti vamp3, by Synaptic Systems (1 mentions)
Anti vamp4, by Synaptic Systems (1 mentions)
Anti vamp8, by Synaptic Systems (1 mentions)
Anti syntaxin2, by Synaptic Systems (1 mentions)
Anti syntaxin 4, by Synaptic Systems (1 mentions)
Anti syntaxin 5, by Synaptic Systems (1 mentions)
Anti snap23, by Synaptic Systems (1 mentions)
Anti glut1, by Abcam (1 mentions)
Ab24170, by Abcam (1 mentions)
Anti tfr, by Thermo Fisher Scientific (1 mentions)
Ab193349, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti snap25, by Abcam (1 mentions)
Anti munc18, by Synaptic Systems (1 mentions)
Curcumin, by Merck Group (1 mentions)
11 protocols using anti vamp2
1
Antibody Characterization for GLUT and SNARE
2
Quantifying Presynaptic Protein Colocalization
3
Immunofluorescence Labeling of Endocytic Organelles
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Cells were enzymatically labeled, fixed in 2% paraformaldehyde, 7% sucrose, in PBS. Free aldehydic groups from paraformaldehyde were covered incubating with 50 mM NH4Cl for 10 min. Buffer containing 0.25% Saponin, 5% normal goat serum, and 2% serum bovine albumine in PBS was used for permeabilization and blocking; incubation with antibodies was performed in the presence of 0.25% Saponin. The following primary antibodies were used: mouse antiearly endosome antigen 1 (anti-EEA1) 1:100 (610456, BD Biosciences, San Jose, CA, USA); rabbit antilysosomal-associated membrane protein 1 (anti-LAMP1) 1:500 (ab24170; abcam, Cambridge, UK); rabbit anti-Rab4 1:100 (sc-312, Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-Rab5 1:100 (sc-28570, Santa Cruz Biotechnology); rabbit anti-Rab7 1:100 (sc-10767, Santa Cruz Biotechnology); rabbit anti-Rab11 10 µg/ml (71-5300, Invitrogen); mouse antivesicle-associated membrane protein 2 (anti-VAMP2) 1:500 (Synaptic Systems, Gottingen, Germany); rabbit anti-VAMP3 1:200 (ThermoFisher Scientific); rabbit anti-Syntaxin-6 1:100 (Synaptic Systems); and mouse antitransferrin receptor (anti-TfR) 1:200 (Invitrogen). The secondary antibodies used were: goat anti-Mouse Alexa Fluor® 405 conjugate 1:250 (Invitrogen); goat anti-Rabbit DyLight 405 conjugate 1:500 (ThermoFisher Scientific). Immunofluorescence microscopy was performed as described before.
4
Western Blot Analysis of Synaptic Proteins
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Total protein samples for western blot analysis were extracted from cultured NRNs or the left ventricle and hippocampi of the mice after they were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.). Mouse death was then confirmed by exsanguination according to a previously described method [31 (link)]. Hippocampi for primary cell culture were collected from neonatal Sprague-Dawley (SD) rats after the administration of 20% isoflurane and confirmation of death by cervical dislocation. Anti-SNAP-25 (1:1000, ab5666, Abcam, MA, USA), anti-VAMP-2 (1:10000, 104,211, Synaptic Systems, Gottingen, Germany) and anti-Syntaxin-1A (1:5000, 100,111, Synaptic Systems, Gottingen, Germany), anti-Munc-18 (1:1000, 116,002, Synaptic Systems, Gottingen, Germany), anti-CD63 (1:1000, ab193349, Abcam, MA, USA),were used as primary antibodies. β-actin (1:1000, G8795, Sigma, Saint Louis, MO, USA) was selected as an internal control. The blots were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The final results were expressed as fold changes compared with the control values.
5
Neuronal SNARE Protein Inhibitors
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Cytosine β-D-arabinofuranoside hydrochloride (C6645), DNase I from bovine pancreas (DN25), poly-L-lysine hydrobromide (P1274) and trypsin (T4799) were purchased from Sigma Aldrich (St. Louis, Missouri, USA).
Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was purchased from Abcam(Cambridge, UK), while anti-syntaxin-1A (STX-1A, 110111) and anti-VAMP-2 were purchased from Synaptic System (Göttingen, Germany). Secondary antibodies: HRP-conjugated Ab were purchased from Calbiochem® (San Diego, CA, USA), Alexa Fluorophores 488- or 555-conjugated Ab were from Thermo-Fisher Scientific (Waltham, MA, USA). Native TeNT was purified as previously described [58 (link)].
Inhibitors: myricetin [3,3′,4′,5,5′7-Hexahydroxyflavone] and Curcumin [(E,E)-1,7-bis(4-Hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione] were purchased from Sigma Aldrich. PX12 [2-[(1-Methylpropyl) dithio]-1H-imidazole] and Ebselen [2-Phenyl-1,2-benzisoselenazol3(2H)-one] were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 4-Bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) was synthesized as in [47 (link)].
Primary antibodies: anti-SNAP-25 (SMI81, ab24737) was purchased from Abcam(Cambridge, UK), while anti-syntaxin-1A (STX-1A, 110111) and anti-VAMP-2 were purchased from Synaptic System (Göttingen, Germany). Secondary antibodies: HRP-conjugated Ab were purchased from Calbiochem® (San Diego, CA, USA), Alexa Fluorophores 488- or 555-conjugated Ab were from Thermo-Fisher Scientific (Waltham, MA, USA). Native TeNT was purified as previously described [58 (link)].
Inhibitors: myricetin [3,3′,4′,5,5′7-Hexahydroxyflavone] and Curcumin [(E,E)-1,7-bis(4-Hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione] were purchased from Sigma Aldrich. PX12 [2-[(1-Methylpropyl) dithio]-1H-imidazole] and Ebselen [2-Phenyl-1,2-benzisoselenazol3(2H)-one] were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 4-Bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone (EGA) was synthesized as in [47 (link)].
6
Western Blot Analysis of Neuronal Proteins
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Primary neurons were lysed on DIV14 using 2% SDS in Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl, pH 7.4) containing phosphatase and protease inhibitors. Cells were scraped and sonicated for a total of 10 s (1 s on/off pulse, 30% amplitude). Lysates were centrifuged at 20,000 g, and the supernatant was diluted into 4X Laemmli buffer with 10% dithiothreitol. Protein lysates were electrophoresed on 8% (LRRK2) or 15% (α-synuclein) SDS-PAGE gels and transferred overnight onto PVDF membranes (Millipore). Blots were either blocked with Everyblot blocking reagent (BioRad) for LRRK2 and phospho-protein blots, or 5% milk in TBS-Tween followed by primary antibody incubation in blocking buffer overnight at 4 °C. The following antibodies were used: anti-LRRK2 c42-2 (Abcam, RRID: AB_2713963), anti-LRRK2 pS1292 (Abcam, RRID: AB_2732035), anti-LRRK2 pS935 (Abcam, RRID: AB_2732035), anti-ɑβsynuclein (Abcam, RRID: AB_869971), anti-vGLUT1(Synaptic Systems, RRID: AB_887878), anti-VAMP2 (Synaptic Systems, RRID: AB_887811), anti-dopamine transporter N-terminus rat monoclonal (RRID: AB_2190413), and anti-vinculin (Biorad, RRID: AB_2214389). Blots were then incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch) for 2 h at room temperature and developed with ECL (Biorad 40–720-71KIT). Blots were quantified using FIJI software.
7
Presynaptic Protein Co-localization Assay
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Immunocytochemistry was performed following paraformaldehyde fixation of cells and permeabilization with 0.1% Triton X-100. For presynaptic co-localization assays, anti-bassoon at 1:500 (Abcam, #ab82958) and anti-VAMP2 at 1:1,000 (Synaptic Systems, #104202) was used to stain thepresynaptic terminal. The number of GFP puncta positive for bassoon or VAMP2 were counted and reported as a percentage of total GFP puncta. Each field is counted as one n and consists of at least 20 puncta and at least 3 independent neuronal culture preparations were assessed. For quantification in the knockdown-rescue experiments, anti-SynI at 1:500 (Novua # NB300-104) was used to assess SynI levels in mCherry-expressing neurons. All quantification was performed using ImageJ software.
8
Immunocytochemical Detection of Synaptic Markers
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Immunocytochemical stainings were performed as described previously16 (link)37 (link). The following primary antibodies were used: Anti-VAMP2 (rabbit polyclonal, 1:2000, Abcam Cat. No. 3347); Anti-N-cadherin (rabbit polyclonal, 1:500–1:2000, Abcam Cat. No. 18203); Anti-PSD95 (mouse monoclonal, 1:400–1:1000, Abcam Cat. No. 2723); Anti-VAMP2 (guinea pig polyclonal, 1:500, Synaptic Systems Cat. No. 104204).
9
Immunofluorescence Staining of Mouse Synaptic Proteins
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We used the following mouse monoclonal antibodies: anti-Bassoon (Enzo, Life Sciences cat. no.: O88778); anti-Synapsin, anti-Synaptophysin, anti-Synaptotagmin-1 (Synaptic Systems cat. no.: 106001, 101011, 104202); anti-TGN38 (BD Biosciences cat. no.: 610899). In addition we used the following rabbit polyclonal antibodies: anti-GFP 6556 (Abcam cat. no.: ab6556); anti-MAP2, anti-Mover, anti-TRIM 36, anti-VAMP2, (Synaptic Systems cat. no.: 188004, 248003, 377003, 104203); anti-Rogdi, directed against amino acids 14–95 of human Rogdi (Sigma-Aldrich cat. no.: hpa041000); anti-Rogdi, directed against full-length human Rogdi GST-fusion protein (Proteintech cat. No.: 17047–1-AP), validated by knockdown10 (link). In addition, we used the following guinea pig polyclonal antibodies: anti-MAP2, anti-Neurofilament H (Synaptic Systems cat. no.: 188004, 171104).
10
Western Blot Analysis of SNARE Proteins
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Cells were directly lysed with Laemmli sample buffer supplemented with protease inhibitors (Roche). Cell lysates were loaded onto NuPage 4–12% Bis-Tris gels (Life Technologies) and separated by electrophoresis in MOPS buffer. Proteins were transferred onto Protran nitrocellulose membranes (Whatman) and saturated for 1 h in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% non-fatty milk. anti-SNAP25 (SMI-81, 1:10,000) was from Biologend; anti-VAMP-2 (104211, 1:2000) was from Synaptic System; anti-SNAP25 BoNT/A-cleaved (1:5000) was home made and used as previously described71 (link); anti-Syntaxin-1A/1B (1:2000) was home made and used as previously described72 (link). Incubation with indicated primary antibodies was performed overnight at 4 °C. The membranes were then washed three times with PBS-T and incubated with appropriate HRP-conjugated secondary antibodies (Anti mouse 1:5000 and Anti rabbit& mouse Flourescent labeled 1:2000) for 1 h. Membranes were washed three times with PBS and proteins revealed with an Uvitec gel doc system (Uvitec Cambridge).
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