The interaction between extracellular α-Syn (oligomer and monomer) and P2X7R was assessed as previously described4 (link). Briefly, precleaning was performed by the addition of 50 μl rProtein G agarose beads (Thermo Fisher, UK) and 2 μg anti-P2X7R, anti-human α-Syn, or species-relevant nonspecific mouse or rabbit immunoglobulin G (IgG). After 4 h of rotation at 4 °C, the beads were centrifuged at 1000 × g for 3 min and then washed with PBS 4 times. Primary microglia were harvested and lysed with co-IP lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.5% Triton X-100) supplemented with 1:100 Halt protease and phosphatase inhibitor cocktail (Roche, Switzerland). The lysates were rotated at 4 °C for 30 min and cleared by centrifugation at 12,000 × g. Samples were then incubated with antibody-conjugated beads overnight at 4 °C. The proteins were separated by SDS-PAGE (10–12%) and electrotransferred onto polyvinylidene fluoride membranes. Western blot analysis was carried out as described above.
Halt protease and phosphatase inhibitor cocktail
Manufactured by Roche
Sourced in Switzerland, China, United States
Halt Protease and Phosphatase Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases and phosphatases in biological samples. It is used to prevent the degradation of proteins and the dephosphorylation of phosphoproteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Synaptophysin, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
0.2 μm nitrocellulose, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti gadph, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab7260, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Sc3223, by Santa Cruz Biotechnology (1 mentions)
5 protocols using halt protease and phosphatase inhibitor cocktail
1
Microglia α-Synuclein and P2X7R Interaction
2
Western Blot Analysis of Protein Expression
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Tissue and cell lysates were prepared using radioimmunoprecipitation assay buffer (Sigma‐Aldrich, St. Louis, MO) with 1% Halt Protease and Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN). Protein samples were separated on NuPAGE 4% to 12% Bis‐tris gels (Life Science Technologies) and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat milk in PBS containing 0.05% Tween 20 and probed with antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (Cox2; Santa Cruz Biotechnology, Santa Cruz, CA), ABCD1, C1q, postsynaptic density protein 95 (PSD95), and CD11b (Abcam, Cambridge, MA), synaptophysin (Cell Signaling Technology, Danvers, MA), MFGE8 (MBL International Corporation, Woburn, MA), IBA1 (Wako Chemicals USA, Inc.), beta‐actin or glyceraldehyde‐3‐phosphate dehydrogenase (Santa Cruz Biotechnology) were used as protein loading control. Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA) after incubation with horseradish peroxidase–conjugated secondary antibodies. Antibodies that differed in human specimen included anti‐human MFGE8 (Sigma‐Aldrich) and anti‐human ABCD1 (Origene, Rockville, MD).
3
Western Blot Protein Quantification
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Cells were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Roche) and total protein concentrations were determined with a BCA Protein Assay Kit (Thermo Scientific). 10 μg total protein/sample was loaded into precast 4–12 % bis-tris gels and run with MES buffer (Invitrogen). Gels were transferred onto 0.2 μm nitrocellulose (BioRad) and incubated with antigen-specific primary antibodies at 4 °C overnight. For LiCor detection, membranes were incubated with species-specific IR Dye antibodies (LiCor) and scanned on an Odyssey Infrared Imager. Bands were quantified using ImageJ software (version 1.48, NIH).
4
Western Blot Analysis of Notch Pathway
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Cells were harvested using RIPA lysis buffer (#P0013B, BiYunTian, Shanghai, China) in the presence of halt protease and phosphatase inhibitor cocktail (#4906845001/#4693116001, Roche, Basel, Switzerland). Western blot analysis was performed as previously described. In brief, equal amounts of proteins were resolved by SDS-PAGE and then transferred to nitrocellulose membranes. The resulting membranes were incubated with primary antibodies at 4ºC overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 hr. The immunoreactive bands were visualized by enhanced chemiluminescence. Primary antibodies used in this study were: Jagged-1 (#70109, CST, Boston, MA, USA), NICD (#3608, CST, Boston, MA, USA), HES1 (#11988, CST, Boston, MA, USA), VEGFR2 (#9698, CST, Boston, MA, USA), β-actin (#4970, CST, Boston, MA, USA).
5
Western Blot Analysis of Neural Proteins
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Tissue and cell lysates were prepared by using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) with 1% Halt Protease and Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN, USA). Protein samples were separated on NuPAGE 4–12% Bis-tris gels (Invitrogen, Carlsbad, CA, USA) and transferred on PVDF membranes. Membranes were blocked with 5% non-fat milk in PBS containing 0.05% Tween 20 and probed with antibodies against ABCD1 (Abcam, ab197013, 1:5000), GFAP (Abcam, ab7260, 1:1000), CGRP (Santa Cruz, sc57053, 1:500) and peripherin (Merke, MAB1527, 1:500). Anti-β-ACTIN (Santa Cruz, SC-47,778, 1:1000) and anti-GADPH (Santa Cruz, sc3223, 1:1000) were used as a protein loading control. Membrane protein signals were prepared by using SuperSignal West Pico Chemiluminescent Substrate (Thermo, Rockford, IL, USA) after incubation with HRP-conjugated secondary antibodies.
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