For total protein extraction and western blot analysis, cells were lysed in ice-cold RIPA buffer [50 mM Tris-Cl (pH 8.0), 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl] supplemented with protease inhibitors (Roche). The lysate was incubated on ice for 30 min and sonicated with ice-cooling for 3 × 5 s pulses at a frequency of 5 microns using a Soniprep 150 plus (MSEs) followed by centrifugation at 13,000 × g for 10 min at 4°C. The amount of protein was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). Typically, 50 μg of total protein in Laemmli buffer [2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8), bromophenol blue 0.02%, 1% β-mercaptoethanol] was heated to 95°C for 5 min and separated by SDS-PAGE and subsequently transferred to Amersham Protran 0.2 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% non-fat dry milk in TBST [20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and probed with indicated primary antibodies overnight at 4°C. Membranes were then washed with TBST three times for 10 min at room temperature followed by incubation for 1 h with the appropriate secondary antibody. The membrane was washed again three times and signals were detected using ECL Western Blot Substrate (BioRad) and the luminescent image analyser LAS-4000 (Fujifilm).
Amersham protran 0.2 μm nitrocellulose membrane
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham Protran 0.2 μm nitrocellulose membrane is a laboratory equipment product designed for use in various scientific applications. It serves as a matrix for the transfer and immobilization of proteins, DNA, and other biomolecules during analytical procedures. The membrane has a pore size of 0.2 micrometers, which facilitates the efficient capture and retention of these target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bcip nbt color development substrate, by Promega (2 mentions)
Chameleon duo pre stained protein ladder, by LI COR (2 mentions)
Anti rabbit irdye 680 cw, by LI COR (2 mentions)
Anti rabbit irdye 800cw, by LI COR (2 mentions)
Anti goat irdye 680 cw, by LI COR (2 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Ecl western blot substrate, by Bio-Rad (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Mini protean tetra vertical electrophoresis cell, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Lide 210 scanner, by Canon (1 mentions)
Complete protein inhibitor cocktail, by Roche (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pa5 21077, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
7 protocols using amersham protran 0.2 μm nitrocellulose membrane
1
Total Protein Extraction and Western Blot Analysis
2
Western Blot Protein Detection
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To prepare whole-cell lysates, cells were lysed in SDS buffer with DTT (Sigma-Aldrich). Proteins were resolved in SDS-PAGE gels with a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, Inc.) at a constant voltage (80–120 V) and transferred to an Amersham Protran 0.2-μm nitrocellulose membrane (GE Healthcare) using semidry or wet systems from Bio-Rad. The membranes were blocked with 5% dried milk in PBS supplemented with 0.5% Tween 20 (Sigma-Aldrich) and then probed overnight with primary antibodies at 4°C followed by alkaline phosphatase–conjugated secondary antibodies for 1 hour at room temperature. Proteins were detected using BCIP/NBT color development substrate (Promega) and dried membranes were scanned using a LiDE 210 scanner (Canon Inc.).
3
Western Blot Protocol Optimization
4
Outer Membrane Protein OmpU Detection
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Expression of OmpU, which is the porin protein in the outer membrane, served as a marker for BEV containing the outer membrane portion. After TCA precipitation, dried pellet was suspended in 30 μl of phosphate-buffered saline (PBS) and 10 μl of 4× Laemmli’s sample buffer and boiled for 5 min. Then the preparation was subjected to 12% SDS polyacrylamide gel and transferred to a Amersham Protran 0.2 μm nitrocellulose membrane (GE Healthcare UK Ltd., UK). Membranes were blocked with 3% skim milk in Tris-buffered saline with Tween 20 (TBST; 150 mM NaCl, 50 mM Tris-HCl, and 0.1% Tween 20) for 30 min before being incubated overnight at 4ºC with rabbit anti-OmpU antibody [17 (link)] (1:1,000 in blocking solution). After incubation with the primary antibody, membranes were washed three times in TBST. Subsequently, goat anti-rabbit IgG-AP (GeneTex, USA) (1:1,000 in blocking solution) was treated as a secondary antibody for 30 min. Then, membranes were washed four times in TBST. Expression of OmpU was visualized using the NBT/BCIP color development substrate (Promega, USA).
5
Western Blot Analysis of Cellular Proteins
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Cells were collected after treatment and lysed in a buffer of 50 nM Tris-HCl, pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.5% Triton X-100 and 1% SDS and freshly supplemented with protease inhibitors (Roche, 11873580001). Then, 15 μg whole lysates per lane and a Chameleon Duo pre-stained protein ladder (LI-COR, 928-60000) were resolved on 4–12% Bis-Tris gels (NuPAGE Invitrogen, NP0321BOX) and transferred to Amersham Protran 0.2-μm nitrocellulose membranes (GE Healthcare, 10600001). Blots were blocked with a LI-COR blocking buffer and incubated with the primary antibodies, namely anti-FTH1 (3998S, Cell Signaling), anti-β-actin (A5441, Sigma), anti-CD71/TfR (Cell Signaling, D7S5Z, 1:1,000 dilution), anti-ZIP14 (PA5-21077, Thermo Fisher, 1:1,000 dilution) and anti-GAPDH (ab9485, Abcam, 1:2,000 dilution) at 4 °C overnight and subsequently incubated with the corresponding secondary anti-IgG antibodies, anti-rabbit IRDye 680 CW (1:15,000 dilution, LI-COR, 926-68071), anti-rabbit IRDye 800 CW (1:15,000 dilution, LI-COR, 926-32211) and anti-goat IRDye 680 CW (1:10,000 dilution, LI-COR, 926-68074) for 1 h at room temperature. Blots were analyzed with an Odyssey Fc imaging system (LI-COR). For densitometry, OD of the protein of interest relative to the housekeeping gene was normalized to the averages obtained in control samples.
6
Protein Detection in Cellular Samples
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To detect proteins in supernatants or cell lysates, 44 µl of supernatant was mixed with 11 µl of non-reducing lane marker sample marker (Thermo Fisher Scientific) while 20 µl of cell lysate was mixed with 5 µl reducing protein sample marker. Supernatants or lysates were then boiled for 5 min before loading 40 µl of supernatant or 20 µl of lysates onto 12% SDS-PAGE gels. After electrophoresis proteins were transferred to Amersham Protran 0.2 μm nitrocellulose membranes (GE Healthcare Life Science, Marlborough, MA). Membranes were blocked with 5% skim milk (Difco, Franklin Lakes, NJ) in 0.05% TBS-T and incubated with a specific primary antibody followed by appropriate secondary antibody conjugated HRP (Table S3). Chemiluminescence signal was developed using Immobilon Forte Western HRP substrate (Merck Millipore, Burlington, MA), and signal was collected using an AZURE c400 Gel Imaging System (Azure Biosystems, Inc., Dublin, CA) or X-ray film. Protein band intensity was analyzed using the Image J program version 1.47 [12 ].
7
Western Blot Analysis of Cell Signaling
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Cells were harvested after treatment with the indicated compounds in NET buffer (150 mM NaCl, 50 mM Tris pH 8, 2 mM EDTA, 1% Triton, 0.1% FBS). Identical amounts of whole lysates (30 μg) together with a Chameleon™ Duo pre‐stained protein ladder (LI‐COR, #928‐60000) were resolved in 4–12% Bis‐Tris gels (NuPAGE Invitrogen, #NP0321BOX) and transferred to Amersham Protran 0.2 μm nitrocellulose membranes (GE Healthcare, #10600001). Blots were incubated with the primary antibodies rabbit anti‐P‐Rb S887/811 (1:1,000, Cell Signaling, #8516), rabbit anti‐FoxM1 D1205 (1:5,000, Cell Signaling, #5436), rabbit anti‐ACTB GTU‐88 (1:10,000, Sigma, #T6557), and goat anti‐p21 C‐19‐G (1:200, Santa Cruz, #sc‐397‐G) at 4°C overnight, and subsequently incubated with the corresponding secondary anti‐IgG antibodies anti‐rabbit IRDye 680 CW (1:15,000, LI‐COR, #926‐68071), anti‐rabbit IRDye 800 CW (1:15,000, LI‐COR, #926‐32211), and anti‐goat IRDye 680 CW (1:10,000, LI‐COR, #926‐68074) for 1 h at room temperature. Blots were analyzed with an Odyssey Fc imaging system (LI‐COR).
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