Gephyrin

Manufactured by BD

Gephyrin is a protein that plays a critical role in the clustering and anchoring of glycine and GABA receptors at inhibitory synapses in the central nervous system. It is essential for the proper localization and function of these neurotransmitter receptors, which are involved in regulating neuronal excitability and inhibitory neurotransmission.

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Lab products found in correlation

γ tubulin, by Merck Group (2 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) β actin, by Merck Group (1 mentions) Cadm1, by Merck Group (1 mentions) Bip grp78, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Synaptophysin, by Merck Group (1 mentions) Glua2 3, by Merck Group (1 mentions) Affi gel columns, by Bio-Rad (1 mentions) Gephyrin, by Synaptic Systems (1 mentions) Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions) Synapsin 1, by Merck Group (1 mentions) Cy3 and alexafluor 488 conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)

4 protocols using gephyrin

Insulin Release Assay in MIN6 Cells

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MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 4.5 g/l glucose supplemented with 15% v/v heat-inactivated FCS, 50 μM β-mercaptoethanol, and 50 mg/ml penicillin, and 100 mg/ml streptomycin and insulin release was performed as described (Poy et al., 2004 (link)). Antibodies were as described: Gephyrin (BD 610584), Rasd1 (Millipore AB15794), Ago2 (for immunoprecipitation, Wako 018-22021), Ago2 (for western blotting, Cell Signaling C34C6), Ago1 (MBL RN028PW), Slc25a22 (Sigma AV44041), HuD/Elavl4 (Santa Cruz sc-48421), γ-tubulin (Sigma T6557), Cadm1 (Sigma S4945), and β-actin (Sigma). Guinea pig anti-insulin and rabbit anti-glucagon antibodies (Millipore) were used on paraffin-embedded pancreata fixed in 4% paraformaldehyde for 3 hr.

Tattikota S.G., Rathjen T., McAnulty S.J., Wessels H.H., Akerman I., van de Bunt M., Hausser J., Esguerra J.L., Musahl A., Pandey A.K., You X., Chen W., Herrera P.L., Johnson P.R., O’Carroll D., Eliasson L., Zavolan M., Gloyn A.L., Ferrer J., Shalom-Feuerstein R., Aberdam D, & Poy M.N. (2014). Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell. Cell Metabolism, 19(1), 122-134.

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Pancreatic Protein Expression Analysis

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The following primary antibodies were used for Western blots at 1:1000 dilution: PITPNA (Abcam, ab180234), Cadm1 (MBL, CM004-3), Gephyrin (BD Biosciences, 610585), CHOP (Cell Signaling, 2895S), BiP/GRP78 (Cell Signaling, 3177S), DRP1 (Proteintech, 12957-1-AP), β-Actin (Cell Signaling, 3700S), and γ-Tubulin (Sigma, T6557). The following primary antibodies were used for immunofluorescence: PITPNA (1:200, Sigma, SAB1400211). Antibodies were used on paraffin-embedded pancreata fixed in 4% paraformaldehyde for 3 h. Image densitometry of 16-bit TIF images for all western blots was performed using ImageJ. All original uncropped western blot images are presented in Supplementary Figs. 7–13.

Yeh Y.T., Sona C., Yan X., Li Y., Pathak A., McDermott M.I., Xie Z., Liu L., Arunagiri A., Wang Y., Cazenave-Gassiot A., Ghosh A., von Meyenn F., Kumarasamy S., Najjar S.M., Jia S., Wenk M.R., Traynor-Kaplan A., Arvan P., Barg S., Bankaitis V.A, & Poy M.N. (2023). Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction. Nature Communications, 14, 4250.

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Antibody Dilutions for Western Blot and Immunostaining

Cited 2 times

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The following antibodies were used with indicated dilutions: rabbit polyclonal antibodies to GABA_AR α1 (1:3,000 for IB), γ2 (1:2,000 for IB), α6 (1:1,000 for IB), GluA2/3 (1:1,000 for IB) (Millipore), GABA_AR γ2 (1:2,000 for ICC, 1:500 for IHC), Neuroligin-2 (1:3,000 for IB, 1:200 for ICC) (Synaptic systems), rabbit normal IgG (Santa Cruz), Neto2 (0.1 μg/mL for IB) (Zhang et al., 2009 (link)), α2 (1:500 for IB) (Abcam) and α3 (1:500 for IB) (Novus Biologicals): mouse monoclonal antibodies to α1 (1:2,000 for IB), PSD-95 (1:2,000 for IB, 1:1,000 for ICC and IHC) (NeuroMab), β2/3 (1:1,000 for IB), Actin (1:5,000 for IB) (Millipore), NR1 (1:2,000 for IB, 1:500 for IHC), Gephyrin (1:1,000 for IB), GAD65 (1:1,000 for IHC) (BD Biosciences), Gephyrin (1:1,000 for ICC) (Synaptic Systems), Synaptophysin (1:5,000 for IB), Tubulin (1:5,000 for IB), FLAG (1:1,000 for IB) (Sigma), HA (1:1,000 for IB) (Covance): guinea pig polyclonal antibodies to GFP (0.1 μg/mL for IB, ICC and IHC) (Kim et al., 2010 (link)). Polyclonal antisera to Lhfpl4 proteins (GARLH4) were raised by injecting rabbits with a GST-LH4 fusion protein encoding last 52 amino acids of LH4. Antisera were affinity purified on Affi-gel columns (Bio-Rad) containing the His-tagged LH4 fusion proteins (0.1 μg/mL for IB).

Yamasaki T., Hoyos-Ramirez E., Martenson J.S., Morimoto-Tomita M, & Tomita S. (2017). GARLH family proteins stabilize GABAA receptors at synapses. Neuron, 93(5), 1138-1152.e6.

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Immunostaining Excitatory and Inhibitory Synapses

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For ICC in vitro preparations, cells were fixed with 4% PFA for 15 min at room temperature and washed in PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. Primary antibodies were incubated overnight at 4 °C in 1% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS. Excitatory synapses were labeled with primary antibodies for PSD-95 (UC Davis, Neuromab, Davis, CA; 1:800) and synapsin1 (Sigma; 1:800). Inhibitory synapses were labeled with primary antibodies for V-Gat (Millipore, Billerica, MA; 1:200) and Gephyrin (BD Bioscience, San Jose, CA; 1:100). Cy3 and Alexa Fluor 488 conjugated secondary antibodies (Jackson ImmunoResearch; 1:500) in 1% normal donkey serum in PBS were applied for 2 hours at room temperature. The nuclear stain (DAPI; 1:20,000) was added prior to mounting coverslips in DABCO. Fluorescent images were obtained with an inverted fluorescence microscope using the Metamorph imaging system.

Weissberg I., Wood L., Kamintsky L., Vazquez O., Milikovsky D.Z., Alexander A., Oppenheim H., Ardizzone C., Becker A., Frigerio F., Vezzani A., Buckwalter M.S., Huguenard J., Friedman A, & Kaufer D. (2015). Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood-brain barrier dysfunction. Neurobiology of disease, 78, 115-125.

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