Total protein was extracted from cells in RIPA Lysis buffer with protease and phosphatase inhibitor cocktail. Total protein amount was quantified by Bradford assay. 15μg of protein was subjected to electrophoresis using NuPAGE Novex Cel System (Life Technologies, Carlsbad, CA) and transferred to nitrocellulose membrane using iBlot Gel Transfer System (LifeTechnologies, Carlsbad, CA) according to manufacturer’s instructions. Anti-hypocretin receptor-1 antibody (ab68718, Abcam, Cambridge, MA) and anti-βactin antibody (clone 13E5, Cell Signaling, Danvers, MA) were used.
Iblot gel transfer system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Hungary, Germany
The IBlot gel transfer system is a laboratory instrument designed for the efficient transfer of proteins from polyacrylamide gels to membranes. The system utilizes an electric current to facilitate the transfer process, allowing for the effective and reliable detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Chemidoc touch imaging system, by Bio-Rad (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase conjugated goat anti rabbit or goat anti mouse polyclonal antibody, by Santa Cruz Biotechnology (3 mentions)
Western lightning ultra chemiluminescent substrate, by PerkinElmer (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Xcell surelock mini cell units, by Thermo Fisher Scientific (3 mentions)
Westernbreeze chromogenic western blot immundetection kit, by Thermo Fisher Scientific (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Las 4000, by Fujifilm (3 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Ecl prime western blotting detection system, by GE Healthcare (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nupage novex cel system, by Thermo Fisher Scientific (2 mentions)
Anti hypocretin receptor 1 antibody ab68718, by Abcam (2 mentions)
Anti βactin antibody clone 13e5, by Cell Signaling Technology (2 mentions)
Novex nupage gel system, by Thermo Fisher Scientific (2 mentions)
84 protocols using iblot gel transfer system
1
Western Blot Analysis of Hypocretin Receptor-1
2
Immunoblot Analysis of IRF3 Activation
3
Western Blot Analysis of PPM1A Protein
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Cells were harvested by centrifugation, washed once with PBS, and lysed in RIPA buffer (Cell Signaling) according to the manufacturer’s instructions. Protein concentration of the lysates was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s recommendations (Thermo Scientific). About 10 to 20 μg of protein per sample was separated on 10% Mini-Protean TGX gels (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Life Technologies). Western blot analysis was performed according to standard protocols. Total PPM1A, α-tubulin, or GAPDH proteins were detected with specific antibodies (see antibody section). A horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibody (Santa Cruz) was used as the secondary antibody. The blot was developed using the Western Lightning Ultra chemiluminescent substrate from Perkin Elmer, Inc., and detected in an EpiChemi3 darkroom (UVP BioImaging Systems).
4
Hippocampal Protein Extraction and Western Blot
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Total hippocampi were lysed in ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 8.0, 10%, 10 mM NaF, 1 mM sodium orthovanadate and protease inhibitors cocktail). Protein extracts were loaded on a SDS–PAGE gel for electrophoresis migration and separated bands were transferred to a polyvinylidene difluoride membrane for 7 min using the iBlot gel transfer system (Life Technologies, St Aubin, France). Nonspecific binding of antibodies was prevented by preincubating the membranes in a blocking buffer (phosphate-buffered saline-Tween 0.01% supplemented with BSA 5%, w/v) for 1 h at room temperature. Then, membranes were incubated overnight at 4 °C in the same blocking buffer supplemented with the primary antibody (see Supplementary Table S4 ). After extensive washing, membranes were subsequently incubated with appropriate fluorescent secondary antibodies (Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 488 donkey anti-mouse; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Finally, membranes were washed, dried and scanned using the Odyssey infrared imaging system (Westburg, Leusden, The Netherlands). The intensity of bands of interest was quantified using ImageJ software. The quantity of proteins loaded on gel was controlled using the housekeeping protein β-tubulin (Cell Signaling Technology, Saint-Quentin-en-Yvelines, France).
5
Western Blot Analysis of Hypocretin Receptor-1
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Total protein was extracted from cells in RIPA Lysis buffer with protease and phosphatase inhibitor cocktail. Total protein amount was quantified by Bradford assay. 15μg of protein was subjected to electrophoresis using NuPAGE Novex Cel System (Life Technologies, Carlsbad, CA) and transferred to nitrocellulose membrane using iBlot Gel Transfer System (LifeTechnologies, Carlsbad, CA) according to manufacturer’s instructions. Anti-hypocretin receptor-1 antibody (ab68718, Abcam, Cambridge, MA) and anti-βactin antibody (clone 13E5, Cell Signaling, Danvers, MA) were used.
6
VEGF-A and FGF-2 Stimulation of HMEC-1 Cells
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HMEC-1 were grown in 6-well titre plates. Cells were serum-arrested at ~80%–90% confluency for 24 h. Cells were then treated with TRAIL (10 ng/mL), VEGF-A (10 ng/mL) and FGF-2 (10 ng/mL) for 15 min, followed by total protein isolation using 1× SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 50 mM dithiothreitol and 0.1% bromophenol blue), as previously described [4 (link)]. Lysates were boiled for 5 min, followed by separation using 10-well Bolt™ 4%–12% Bis-Tris Plus Gels (Thermo Fisher Scientific, Waltham, MA, USA). The iBlot Gel Transfer System (Life Technologies, Carlsbad, CA, USA) was used to transfer proteins onto nitrocellulose membranes. Membranes were blocked in 5% bovine-serum albumin (in 1× PBS-Tween-20) for 1 h and then incubated overnight at 4 °C with rabbit polyclonal phospho-p42/44 MAPK(ERK1/2) antibody (1:5000; Cell Signaling Technology, Danvers, MA, USA) or 42/44 MAPK(ERK1/2) (1:3000; Cell Signaling Technology, Danvers, MA, USA). Proteins were detected with horseradish peroxidase-conjugated secondary anti-rabbit antibodies (1:3000; Dako, Glostrup, Denmark) and visualized by chemiluminescence (Amersham, GE Healthcare Life Sciences, Logan, UT, USA).
7
Virus Lysate Western Blotting Protocol
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Purified HSV-2 lysate (Advance Biotechnologies, Columbia, MD) was resolved in 4 to 12% bis-Tris precast gradient SDS-polyacrylamide gels (Life Technologies) and blotted onto a polyvinylidene difluoride membrane using an iBlot gel transfer system (Life Technologies). Membranes were blocked with 5% bovine serum albumin (Sigma) overnight. The blocked membranes were incubated overnight at 4°C with 1 μg/ml GRFT in PBS or with mouse anti-gD MAb DL11 diluted 1:500 in 5% nonfat instant dry milk, PBS, and 0.1% Tween. The membranes were washed and incubated with rabbit polyclonal anti-GRFT (Pacific Immunology, Ramona, CA) or anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP) (Promega, Madison, WI). After the required washing steps, the membrane used to detect GRFT binding was incubated with anti-rabbit immunoglobulin conjugated with HRP and washed again, and both membranes were processed using an enhanced chemiluminescence Western blotting detection system (Thermo Scientific).
8
Western Blot Analysis of PPM1A Protein
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Cells were harvested by centrifugation, washed once with PBS, and lysed in RIPA buffer (Cell Signaling) according to the manufacturer’s instructions. Protein concentration of the lysates was determined by the bicinchoninic acid (BCA) method according to the manufacturer’s recommendations (Thermo Scientific). About 10 to 20 μg of protein per sample was separated on 10% Mini-Protean TGX gels (Bio-Rad) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer system (Life Technologies). Western blot analysis was performed according to standard protocols. Total PPM1A, α-tubulin, or GAPDH proteins were detected with specific antibodies (see antibody section). A horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibody (Santa Cruz) was used as the secondary antibody. The blot was developed using the Western Lightning Ultra chemiluminescent substrate from Perkin Elmer, Inc., and detected in an EpiChemi3 darkroom (UVP BioImaging Systems).
9
SDS-PAGE Analysis of Wheat Germ Protein
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SDS-PAGE analysis was performed essentially as described previously using a 10–20 % Tris-Tricine/Peptide Gel (Bio-Rad, Hercules, CA, USA) [25 (link)]. Protein was transferred onto the PVDF membrane using the iBlot gel transfer system (Life Technologies). For electrophoresis of protein produced in the wheat germ cell-free system, protein was electrophoresed using a Surelock Mini Cell (Life Technologies, Carlsbad, CA, USA), for 35 min at 200 V. Thioredoxin containing protein bands were visualised by incubation with the anti-thioredoxin primary antibody produced in rabbit (Sigma St. Louis, MO, USA), followed by the anti-rabbit-alkaline phosphatase secondary antibody (Sigma) and exposure to the chromogenic BCIP®/NBT-Blue liquid substrate (Sigma) (Figure 2 B). GSL containing bands were visualised by incubation of the blot with a 1:400 dilution of sera containing Rabbit anti-GSL antibodies (raised in this study), followed by incubation with the secondary antibody Immun-Star™ Goat Anti-Rabbit (GAR)-HRP Conjugate (Bio-Rad) with a 1:100,000 dilution. Following the blots development, light signals were captured on Lumi-Film Chemiluminescent Detection Films (Bio-Rad) (Figure 4 B).
10
Protein Detection and Analysis in Neurodegenerative Disorders
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PrPSc, purified as previously described [75 ], hamster ScBH at a dilution of 10−1, α-synuclein, or Lewy bodies isolated from the brains of a Lewy body dementia patient, as described [76 (link)], were pretreated with saline or BrioHOCl solutions as indicated in the figure legends. Following treatment, samples were diluted with equal volumes of 2X sample buffer (125 mM Tris-HCl pH 6.8, 5% glycerol, 6 mM EDTA, 10% SDS, 0.04% bromophenol blue, 48% urea, 8% 2-mercaptoethanol) and boiled for 5 min. Equal volumes of samples were run on 10% Bis-Tris NuPAGE gels (Invitrogen) and used for subsequent Deep Purple protein stain per manufacturer’s instructions (GE Healthcare) or the protein transferred to an Immobilon P membrane (Millipore) using the iBlot Gel Transfer System (Life Technologies). PrP was detected using rabbit PrP antisera R30 (1:10,000; residues 90–104) [77 (link), 78 ] and alkaline-phosphatase conjugated secondary antibody (1:5000; Jackson ImmunoResearch). α-Synuclein was detected using mouse Anti-α-Synuclein Clone 42 antibody (1:1000; BD Transduction Laboratories) and alkaline-phosphatase conjugated secondary antibody (1:2000; Jackson ImmunoResearch). Tau was detected using a tau antibody (anti-tau ab64193, Abcam) as the primary antibody.
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