EVs were isolated from 10 ml of conditioned medium of 3T3-L1 adipocytes treated with either 0.5 mM palmitic acid or control as described above. The pelleted EVs were then resuspended in 50 µl of RIPA buffer (Cell Signaling, Danvers, MA) with cOmplete proteinase inhibitors (Roche). For circulating EVs, the starting volume of whole blood was 900 µl for mouse and 500 µl for human. Circulating EVs were extracted as described above and resuspended with 200 µl of RIPA buffer with proteinase inhibitors. 25 µl of EVs were mixed with β-mercaptoethanol-reduced 6× sodium dodecyl sulfate buffer (Biomiga, San Diego, CA) and resolved by Criterion™ TGX Any kD™ Precast Gel (Bio-Rad, Hercules, CA). Proteins were transferred to 0.2 µm nitrocellulose membrane (Bio-Rad) and blocked for 1 h with 5% bovine serum albumin in TBS, 0.05% Tween 20. Blots were then hybridized overnight using antibody perilipin A (One World Lab, San Diego, CA or ProSci Inc., Poway, CA), cleaved caspase 3 (D175) (Cell signaling), caspase 3 (Cell signaling), phospo-MYPT1 (T696) (Cell signaling), and MYPT1 (Cell signaling). Secondary antibodies used were anti-rabbit (Cell Signaling; 1:2000) and anti-goat (R&D; 1:2000), respectively. Proteins were visualized by SuperSignal West chemiluminescence substrate (Pierce biotechnology, Rockford, IL). Band intensity was analyzed using Image Lab (Bio-Rad).
Criterion tgx any kd precast gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion™ TGX Any kD™ Precast Gel is a ready-to-use polyacrylamide gel designed for protein electrophoresis. It features a proprietary formulation that allows for the separation of a wide range of protein molecular weights on a single gel.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti histone 3, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Precision plus protein standard, by Bio-Rad (2 mentions)
Complete proteinase inhibitors, by Cell Signaling Technology (1 mentions)
Mypt1, by Cell Signaling Technology (1 mentions)
Anti goat, by R&D Systems (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
0.2 m nitrocellulose membrane, by Bio-Rad (1 mentions)
Cleaved caspase 3 d175, by Cell Signaling Technology (1 mentions)
Anti rabbit, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Supersignal west chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Chameleon duo pre stained protein ladder, by LI COR (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Anti actin, by Merck Group (1 mentions)
6 protocols using criterion tgx any kd precast gel
1
Extracellular Vesicle Protein Analysis
2
FMRP Point Mutation Westernblot
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To analyze the point mutation case, 1016–15, (nominal concentrations of 8 μg and 16 μg total protein) was determined by bicinchoninic acid (BCA) assay (Pierce; Rockford, IL; cat. no. 23225) for each sample and was run on a Criterion™ TGX Any kD™ Precast Gel (BioRad; Hercules, CA; cat. no. 567–1125) alongside 5 μl of Chameleon Duo Pre-stained Protein Ladder (LI-COR; Lincoln, NE; cat. no. 928–60000) (marker) for 30 min at 25 mA and then 1.5 h at 150 V. Proteins were transferred to a nitrocellulose membrane at 30 V overnight at 4°C. The membrane was blocked in 1:1 PBS:Odyssey® Blocking Buffer (LI-COR; 927–40000) for 1 h at room temperature. Mouse anti-FMRP (Millipore; Burlington, MA; cat. no. MAB2160; Lot no. 2548166) primary antibody in blocking buffer, supplemented with 0.1% Tween-20, was applied to the membrane at 1:5,000 overnight at 4°C. Subsequently, IRDye® 800CW Goat anti-Mouse (LI-COR; cat. no. 926–32210; Lot no. C50316-03) secondary antibody in blocking buffer, supplemented with 0.1% Tween-20, was applied to the membrane at 1:20,000 for 1.5 h at room temperature. Membrane was visualized on the LI-COR Odyssey Infrared Imager.
3
Histone and Actin Immunoblotting
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Samples were boiled in a sodium dodecyl sulphate (SDS) buffer containing dithiothreitol (DTT) and resolved by polyacrylamide gel electrophoresis (SDS-PAGE) on a Criterion TGX precast gel (Any-KD; Bio-Rad Laboratories). Proteins were then transferred to a PVDF membrane (Bio-Rad Laboratories) via semi-dry transfer. The membrane was blocked with 5% bovine serum albumin (BSA; Fisher Scientific) in Tris-buffered saline with 0.1% Tween 20 (TBS-T). H3 was detected with anti-histone 3 (Milipore) and HRP–conjugated goat anti-rabbit (Thermo Scientific). Actin was detected with anti-actin (Milipore) and HRP–conjugated donkey anti-mouse (Thermo Scientific) antibodies.
4
Western Blot Analysis of Histone 3
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Samples (protein extracts or plasma) were boiled in a sodium dodecyl sulphate (SDS) buffer containing dithiothreitol (DTT) and resolved by polyacrylamide gel electrophoresis (SDS-PAGE) on a Criterion TGX precast gel (Any-KD; Bio-Rad Laboratories). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories) via semi-dry transfer. Non-specific binding was blocked with 5% bovine serum albumin (BSA; Fisher Scientific) in tris-buffered saline with 0.1% Tween 20 (TBS-T). The membranes were blotted with anti-histone 3 (Milipore) and detected with HRP–conjugated goat anti-rabbit (Thermo Scientific). Finally, the membranes were incubated with enhanced chemiluminescent substrate (ECL; Thermo Fisher Scientific) and imaged with a chemiluminescence imaging systems (Bio-Rad) or developed after exposure onto an X-ray film (Kodak) and digitally scanned.
5
SDS-PAGE Protein Electrophoresis Protocol
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Electrophoresis of proteins (SDS-PAGE) was performed according to Laemmli et al. (Laemmli, 1970 (link)) using a Mini-Protean 3 Gel Electrophoresis Unit (Bio-Rad) and Criterion TGX™ Precast Any kD gel (5671124 Bio-Rad Laboratories, Inc., CA, USA). Samples (OF, OPC and COPC) were dissolved in Laemmli buffer (0.1 M Tris-Tricine, pH 6.8, 2% SDS, 5% β-mercaptoethanol and 0.025% bromophenol blue), boiled for 5 min, loaded onto the gel (10 μL, 20 μg protein/well) and run at 150 kV. Gel was stained using 0.125% Coomassie Brilliant Blue R-250 in 7% acetic acid and 40% MeOH (v/v) solution and stained in 7% acetic acid and 30% EtOH (v/v) solution. As a molecular marker, Precision Plus Protein™ standard (10–250 kDa, Bio-Rad Laboratories Inc., CA, USA) was used.
6
SDS-PAGE Analysis of Protein Samples
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The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was performed according to Laemmli et al. (1970) (link) using a Minin-Protean 3 Gel Electrophoresis Unit (Bio-Rad) and Criterion TGX™ Precast Any kD gel (5671124 Bio-Rad Laboratories, Inc., CA, USA). About 2.5 mg of raw, extruded, cooked, and baked samples was dissolved in 1 mL Laemmli buffer (0.1 M Tris-Tricine, pH 6.8, 2% SDS, 5% β-mercaptoethanol, and 0.025% bromophenol blue), stirred for 1 h, boiled for 5 min, and then centrifuged at 10,000 g for 1 min and loaded onto the gel (20 μL samples, 20 μg protein/well) and run at 150 kV. Gel was stained using 0.125% Coomassie Brilliant Blue R-250 in 7% acetic acid and 40% MeOH (v/v) solution and stained in 7% acetic acid and 30% EtOH (v/v) solution. As a molecular marker, Precision Plus Protein™ standard (10-250 kDa, Bio-Rad Laboratories Inc., CA, USA) was used.
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