Pancreatic tissues were ground in liquid nitrogen and then sonicated (power 20%, 5 seconds on, 5 seconds off, total time 5 minutes) in lysis buffer (2% SDS, 10% glycerol, 10 mM Tris (pH 6.8) and 100 mM DTT). The tissue suspension was then centrifuged for 10 minutes (4°C, 12,000 rpm). The supernatant was removed and boiled for 10 minutes. Protein lysates were resolved on NuPAGE™ 4%‐12% Bis‐Tris Mini Protein Gel (#NP0336BOX; Invitrogen) and transferred to 0.45 μm nitrocellulose membranes (#HATF00010; Merck). Protein samples were normalized to GAPDH. Antibodies utilized are listed in Table 1 . Imaging was taken using LI‐COR Odyssey CLx.
Nupage 4 12 bis tris mini protein gel
Manufactured by Thermo Fisher Scientific
The NuPAGE 4%–12% Bis-Tris Mini Protein Gels are pre-cast polyacrylamide gels used for the electrophoretic separation of proteins. The gels feature a Bis-Tris buffer system and a 4% to 12% gradient for the separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hatf00010, by Merck Group (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Imperial protein stain solution, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 1 2, by Merck Group (1 mentions)
Anti cd81 sc 166029, by Santa Cruz Biotechnology (1 mentions)
Anti cd63 sc 5275, by Santa Cruz Biotechnology (1 mentions)
Anti tgf β, by Cell Signaling Technology (1 mentions)
Anti mouse nxa931, by GE Healthcare (1 mentions)
Amersham ecl western blotting detection kit, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti flag antibody clone m2, by Merck Group (1 mentions)
Nupage reducing agent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated anti mouse antibody, by Merck Group (1 mentions)
8 protocols using nupage 4 12 bis tris mini protein gel
1
Protein Extraction and Western Blotting
2
SDS-PAGE Analysis of Purified Antibodies
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Purified 1D8, B5, and B5×1D8 were prepared in NuPAGE LDS sample buffer (Invitrogen, NP0007). For reducing samples, 2-mercaptoethanol (Sigma-Aldrich, M3148) was added in the sample buffer and heated at 70°C for 10 min. A total of 2.5 μg of each antibody and Novex Sharp Pre-stained Protein Standard (Invitrogen, LC5800) was loaded into the wells of NuPAGE 4-12% Bis-Tris Mini Protein Gel (Invitrogen, NP0321). After electrophoresis, the gel was stained by Imperial Protein Stain solution (Thermo Fisher Scientific, 24615).
3
Protein Isolation and Analysis from EVs, Cells, and Lysates
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Protein was collected from EVs, cells or cell lysates through lysis with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1:100 Protease Inhibitor Cocktail Set III and Phosphatase Inhibitor Cocktail 1 & 2 (Sigma-Aldrich). Protein was quantified using a Pierce BCA Protein Assay Kit (ThermoFisher). Ten μg of protein was separated using NuPAGE 4%–12% Bis-Tris Mini Protein Gels (ThermoFisher) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked in 5% BSA, 1X TBS, and 0.1% Tween-20 at RT for 1 h. Membranes were then incubated overnight at 4°C with the appropriate primary antibody: 1:1000 diluted anti-Alix (sc-53540), anti-Hsp-70 (sc-24), anti-Flotillin-1 (sc-74566), anti-CD81 (sc-166029), anti-CD63 (sc-5275), and anti-GRP 94 (sc-393402) (all from Santa Cruz), and anti-TGFβ (#3711, Cell Signaling). After washing the membranes, they were subsequently incubated with peroxidase-conjugated 1:2000 Anti-Mouse (NXA931, GE Healthcare) or Anti-Rabbit (#7074, Cell Signaling). Detection was performed using Amersham ECL Western Blotting Detection Kit (GE Healthcare) and Chemidoc MP Imaging System (Bio-Rad).
4
Detecting Protein Expression by Western Blot
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For each sample, three leaf discs were harvested 48 hr after agro‐infiltration to detect protein expression levels by western blot. Samples were ground in 100 μl NuPAGE LDS sample buffer with NuPAGE Reducing Agent as per the supplier's instructions (Life Technologies) and centrifuged at 15,700 × g for 10 min, and the supernatant was collected. After heating at 70 °C for 10 min, total protein extracts were separated on NuPAGE 4%–12% Bis‐Tris Mini Protein Gels (Thermo Fisher) and transferred to nitrocellulose membranes (Millipore) for immunoblotting. HA‐tagged proteins were detected using a horseradish peroxidase (HRP)‐conjugated anti‐HA antibody (clone 3F10; Roche), while the Pikp‐1:Flag protein was detected using an anti‐FLAG antibody (clone M2; Sigma‐Aldrich) and a secondary HRP‐conjugated anti‐mouse antibody (Sigma‐Aldrich). The Immobilon Western kit (Millipore) was used for detection.
5
SDS-PAGE Gel Electrophoresis Protocol
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Pre-cast NuPAGE™ 4–12% Bis-Tris mini protein gels (ThermoFisher Scientific), 1.0 mm gel thickness and 10 wells, were used with Invitrogen™ mini gel tanks. Samples were prepared by diluting 15 μL of analyte in 10 μL NuPAGE™ 4X LDS loading buffer (ThermoFisher Scientific) before heating at 95 °C for 5 min on a heating block (HB120-S, Scilogex, Rocky Hill, CT, USA). Hydrogels samples were mixed with loading buffer up to a final volume of 100 μL to help dissolving the gel. After heating, 10 μL of each sample was loaded alongside 5 μL of prestained protein ladder, 10 to 250 kDa (PageRuler™ Plus, ThermoFisher Scientific). All gels were run for 1 h at a constant voltage (120 V) using NuPAGE™ MES SDS running buffer (ThermoFisher Scientific). Gels were stained overnight using Coomassie blue (InstantBlue™, Expedeon, Heidelberg, Germany) and imaged using a compact scanner (CanoScan LiDE 220, Tokyo, Japan).
6
Protein Separation and Detection
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Samples were denatured in 1× SDS–PAGE Loading Buffer (50 mM Tris 6.8, 2% SDS, 0.1% Bromophenol Blue, 10% glycerol, 25 mM DTT) at 95°C for 5 min. Denatured samples were separated on NuPAGE 4-12% Bis-Tris Mini Protein Gels (ThermoFisher Scientific).
For Coomassie Blue Stain, gels were stained with ProtoBlue Safe working solution (National Diagnostics) according to the manufacturer's instructions and imaged on Li-cor Odyssey Fluorescent Imaging System.
For Western Blot, wet transfer was performed onto Immobilon-FL PVDF membrane (Merck) and confirmed by reversible Ponceau S stain. Immunoblotting was performed using primary and secondary antibodies listed inTable 2 and imaged on Li-cor Odyssey Fluorescent Imaging System.
For Coomassie Blue Stain, gels were stained with ProtoBlue Safe working solution (National Diagnostics) according to the manufacturer's instructions and imaged on Li-cor Odyssey Fluorescent Imaging System.
For Western Blot, wet transfer was performed onto Immobilon-FL PVDF membrane (Merck) and confirmed by reversible Ponceau S stain. Immunoblotting was performed using primary and secondary antibodies listed in
7
Immunoblotting Protocol for Protein Analysis
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For immunoblotting, RIPA buffer (150 mM NaCl, 50 mM Tris HCl, pH 8, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail [Roche]) was added to cell fractions or whole cells in lysis buffer for 30 min on ice and pelleted. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated in NuPAGE 4%–12% Bis-Tris mini protein gels (Thermo Fisher Scientific) and electro-transferred onto PVDF membranes using iBlot 2 (Thermo Fisher Scientific). Membranes were blocked with 5% milk in TBS with Tween-20 for 2 h and incubated overnight at 4°C with primary antibodies—anti-ELAVL1 (3A2, sc-5261, Santa Cruz Biotechnology), anti-GAPDH (G8795, Sigma-Aldrich), anti-Lamin A + Lamin C (EPR4100, Abcam), anti-PATZ1 (sc390577, Santa Cruz Biotechnology), and anti-MAP3K5 (SAB4300398, Sigma-Aldrich). Membranes were washed and incubated with horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies (A3682 and A9169, Sigma-Aldrich). After washing, proteins were detected with ECL reagent (Bio-Rad), according to the manufacturer's protocol.
8
Cell Lysis and Protein Extraction Protocol
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Cells were cultured at 37°C in 10 cm dish until 70%–80% confluent. Cells collected with trypLE express were washed in PBS and then lysed in RIPA lysis and extraction buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche). After brief sonication, lysed cell suspensions were rotated at 4°C for 1 h for protein extraction followed by centrifugation at 15000g. Protein concentration was determined with the Bio-Rad DC assay kit (Bio-Rad, # 500-0111), and SDS-PAGE was performed using NuPAGE™ 4%–12%, Bis-Tris, mini protein gels (ThermoFisher Scientific # NP0321BOX) according to manufacturer’s instruction. Western blot transfer was done into nitrocellulose membrane (Thermo Scientific #IB23002) using the iBlot 2 Dry Blotting System (Thermo Scientific).
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