Human insulin (Humulin R) was purchased from Eli Lily. FITC-insulin was purchased from Sigma-Aldrich. Vectastain Universal Elite ABC kit, Carbo-Free blocking solution, and DyLight 649-tomato lectin were purchased from Vector Laboratories. Bovine serum albumin (BSA) was purchased from Rockland Immunochemicals. Phospho-insulin receptor beta (Y1185) antibody was purchased from Bioss (#bs-5453R). Rabbit anti-insulin antibody and horseradish peroxidase conjugated anti-rabbit IgG antibody was purchased from Cell Signaling Technology. Neuro-Chrom pan-neuronal antibody was purchased from Millipore (#ABN2300). Phosphatase inhibitor cocktail was purchased from Research Products International. Complete mini protease inhibitor cocktail was purchased from Roche. SuperBlock blocking buffer, 4,6-diamidino-2-phenylindole (DAPI), IP lysis buffer, ProLong Diamond, and Alexa Fluor 568 goat anti-mouse antibody were purchased from Thermo Fisher. Any KD TGX gels, XT sample buffer, reducing agent, and Clarity Western ECL reagent were purchased from Bio-Rad. All other reagents were purchased from Sigma-Aldrich unless noted.
Reducing agent
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Reducing agent is a chemical compound used in laboratory settings to facilitate the reduction of other chemical species. It functions by donating electrons, thereby decreasing the oxidation state of the target compounds.
Automatically generated - may contain errors
Lab products found in correlation
Xt sample buffer, by Bio-Rad (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Nupage lds sample buffer, by Bio-Rad (2 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
A5441, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Sample loading buffer, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Rockland Immunochemicals (1 mentions)
Carbo free blocking solution, by Vector Laboratories (1 mentions)
Dylight 649 tomato lectin, by Vector Laboratories (1 mentions)
Fitc insulin, by Merck Group (1 mentions)
Rabbit anti insulin antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Any kd tgx gels, by Bio-Rad (1 mentions)
Clarity western ecl reagent, by Bio-Rad (1 mentions)
Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
15 protocols using reducing agent
1
Insulin Signaling Pathway Analysis
2
Western Blot Analysis of Mitochondrial Proteins
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Whole cell extracts of mitochondria, resuspended in XT sample buffer (BioRad) and reducing agent (BioRad), were resolved on Criterion XT 12% Bis–Tris gels (BioRad) in XT Mops running buffer (BioRad). Proteins were transferred to Immuno-Blot polyvinylidene difluoride (BioRad) at 100 V for 1 h. Following transfer, membranes were blocked with 5% (w/v) milk and 0.05% (v/v) Tween-20 in PBS (blocking buffer) for 1 h at room temperature or at 4 °C if longer, with blocking. Following blocking, membranes were briefly washed with PBS with 0.2% (v/v) Tween-20 (PBST) and then incubated with primary antibody in PBST with 0.02% (w/v) sodium azide overnight at 4 °C with blocking. Following three successive 10-min washes with PBST at room temperature, horseradish peroxidase–conjugated secondary antibodies were added and incubated for 45 min at room temperature. The membranes were then washed three times for 10 min with PBST and twice for 10 min with PBS. Immunoreactive bands were visualized using ECL Western Blotting Detection (Amersham) or SuperSignal West Pico PLUS (Pierce). Images were captured with a Fluorchem Q (Cell Biosciences, Inc) or on film. Film was scanned before quantification. Quantitation of bands was performed using ImageJ (http://imagej.nih.gov/ij/ ) and protein expression values were normalized to loading controls.
3
Validating Anti-ISAV Antibody Specificity
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Western Blot was performed to check for specificity of anti-ISAV antibodies, the transfection efficiency and status of infection. TO and EPC cells were transfected and infected as previously described; 0.5 ml ISAV (= 106.25 TCID50/25 µl) was used for TO cells. Cell lysates were prepared from infected and mock-infected cells 2 dpi, using RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100). Aggregates and nuclei were removed by centrifugation (1150 g) for 5 min. Protein concentration was measured using Nanodrop. Samples were boiled in SDS sample buffer (Biorad) with reducing agent (Biorad). A total of 160.0 µg protein was loaded per well in precast 4–12% Bis–Tris polyacrylamide gels (Biorad) and separated by electrophoresis using XT-MOPS as running buffer (Biorad). After electroblotting onto a PVDF membrane (Biorad), the proteins were detected either with anti-ISAVs8ORF2 antiserum, anti-penta-his Mab or anti-ISAV antiserum (Aquatic Diagnostics, Stirling, UK) using HRP-conjugated secondary antibodies (GE healthcare) and ECL Plus Western Blotting Detection System (GE Healthcare, UK). The image was captured using Chemidoc XRS (Biorad).
4
Parasite Pellet Preparation and Protein Analysis
5
Quantifying AKT and mTOR Signaling
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WB was performed as previously described [50 (link)] using antibodies against total AKT (rabbit monoclonal - AB179463; Abcam, UK) p-AKT (Thr308) (rabbit monoclonal - 2965, Cell Signalling, UK), p-AKT (Ser473) (mouse monoclonal - 4051, Cell Signalling, UK), total mTOR (rabbit monoclonal - AB134903; Abcam, UK) and p-mTOR (Ser2448) (mouse monoclonal - 5536, Cell Signalling, UK). 50 μg of whole cell protein lysates in RIPA buffer were mixed with NuPAGE® LDS Sample Buffer (BioRad, UK) and reducing agent (BioRad, UK) and denatured at 90 °C for 10 min.
Protein samples were separated on either 8% or 10% polyacrylamide gels depending on their molecular weights and transferred onto PVDF membranes (Millipore, UK). Membranes were next blocked with 5% powder milk in Tris Buffer Saline/0.1% Tween-20 and incubated overnight with primary antibodies, followed by secondary peroxidase conjugated antibody (Fisher Scientific, UK) for 1 h at room temperature. Protein detection was done with Immobilon Western Chemiluminescent HRP Substrate (Millipore, UK). β actin levels were used as a loading control (mouse monoclonal - A5441, Sigma-Aldrich, UK).
Protein samples were separated on either 8% or 10% polyacrylamide gels depending on their molecular weights and transferred onto PVDF membranes (Millipore, UK). Membranes were next blocked with 5% powder milk in Tris Buffer Saline/0.1% Tween-20 and incubated overnight with primary antibodies, followed by secondary peroxidase conjugated antibody (Fisher Scientific, UK) for 1 h at room temperature. Protein detection was done with Immobilon Western Chemiluminescent HRP Substrate (Millipore, UK). β actin levels were used as a loading control (mouse monoclonal - A5441, Sigma-Aldrich, UK).
6
Western Blot Analysis of Protein Signaling
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Cell lysate samples were generated using RIPA buffer supplemented with phosphatase and protease inhibitors (50 mM Tris-HCl pH 7.4, 1% (v/v), 150 mM, NaCl, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA, 0.1% (w/v) SDS, 1 mM Na3VO4, 1 mM PMSF) and protease inhibitor cocktail (Sigma). Protein samples were denatured using sample buffer containing reducing agent (BioRad, hercules, CA, USA) and heated at 70°C for 10 min. Samples were separated on a 10% polyacrylamide gel and subsequently transferred to PVDF membrane (BioRad). The membrane was then blocked in 5% nonfat milk in TBS-T (PBS with 0.1% Tween20) for 1 h at room temperature followed by incubation with primary antibodies of MMP-9 (Abcam, Cambridge, MA, USA), phospho-MAPKAPK2 (CST), NF-кβ-p65 (CST), and β-actin (Santa Cruz, Dallas, TX, USA), overnight at 4°C. The membrane was then washed three times in TBS-T followed by a 1-h room-temperature incubation with IRDye 680RD goat antimouse IgG (LI-COR, Lincoln, NE, USA) secondary antibody. Followed by the incubation, membrane was washed three more times with TBST. Images were captured using Licor Odyssey CLx Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). Abundance was determined by standard densitometry analysis, using ImageJ software (NIH, Bethesda, MD, USA) with β-actin as normalizing protein.
7
Western Blot Analysis of Protein Expression
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Sample loading buffer (Bio-Rad XT sample buffer) and reducing agent (Bio-Rad) was added to the samples according to the manufacturer’s instruction and equivalent amounts of protein (50 µg as measured using the DC Protein Assay Kit (Bio-Rad) and proteins were separated on a 4 to 12% on a 12% gradient XT precast Criterion gel using XT-MOPS buffer (Bio-Rad) at 150–200 V. Subsequently, proteins were transferred onto a PVDF membrane. Membranes were blocked for 30 min in a 1:1 Tris-buffered saline (TBS)/Odyssey blocking solution (cat n° 927-40003, LI-COR, Lincoln, NE, USA) and probed by Western blotting. Following overnight incubation of primary antibody in TBS-T/Odyssey blocking buffer and three 10 min washes in TBS-T (0.1% Tween-20), membranes were incubated with secondary antibody for 30 min in TBS-T/Odyssey blocking buffer followed by 3 washes in TBS-T or TBS (last washing step). The following antibodies were used: mouse anti-GAPDH (Abcam, ab9484, 1:10,000), rabbit purified IgG anti-NAA10 (anti-hARD1, Biogenes GmBH [58 (link)], 1:1000) and anti-NAA15 (anti-NATH, Biogenes GmBH [58 (link)], 1:1000), anti-mouse (IRDye 800 CW goat anti-mouse antibody IgG, LI-COR, cat n°926-32210, 1:10,000) and anti-rabbit (IRDye 800 CW goat anti-rabbit IgG, LI-COR, cat n°926-3221, 1:10,000). The bands were visualized using an Odyssey infrared imaging system (LI-COR).
8
Western Blot Analysis of Flag-tagged Proteins
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The beads-antibody-protein complex was diluted in Sample Buffer (Bio-Rad, Hercules, CA, USA) and Reducing Agent (Bio-Rad), denatured for 5 min at 95 °C and run in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), using 4-12% Bis–Tris Criterion XT gel (Bio-Rad). Lysates from non-transfected EPC cells were used as a negative control, and Precision Plus Protein Western C Standards (Bio-Rad) as a molecular size marker. Following SDS-PAGE, the proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and incubated with primary antibody (anti-flag 1:1000) at 4 °C overnight. After incubation with secondary antibody (Anti-mouse IgG-HRP, GE Healthcare, Buchinghamshire, UK), the proteins were detected by chemiluminescense using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).
9
Immunoprecipitation and Protein Analysis
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Cells were lysed with ice-cold immunoprecipitation buffer [10 mM tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 60 mM n-octyl-β-d -glucopyranoside (Sigma-Aldrich)] containing protease and phosphatase inhibitors. Protein concentrations from cells were determined using Quick Start Bradford Protein Assay reagent (Bio-Rad). Protein (3 to 4 mg/ml) in immunoprecipitation buffer was incubated with control rabbit immunoglobulin G (10 μg/ml; Thermo Fisher Scientific) or monoclonal antibodies to flotillin-1 (ab133497, Abcam) or MMP14 (ab51074, Abcam) overnight on a vertical rotator at 4°C. After, protein G agarose beads (100 μl/ml; Roche) were added and incubated for a further hour. Bound protein complexes were spun down, washed twice with immunoprecipitation buffer and once with 1× TBS, and eluted by heating in 4× Laemmli sample buffer (Bio-Rad) containing reducing agent (Bio-Rad) for 10 min at 95°C for analysis by Western blotting.
10
Western Blot Analysis of Protein Samples
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