For protein extraction, cell pellets were resuspended in RIPA buffer (Sigma #R0278) containing protease inhibitors (Roche #4693159001), incubated at 4°C for 30 min and, upon centrifugation, cell lysis supernatant was collected. 10–20 μg of protein was mixed with bolt LDS sample buffer (ThermoFisher #B0007) and bolt‐reducing agent (ThermoFisher #B0004), heated at 70°C for 10 min and loaded on 4–12% Bis‐Tris gel (ThermoFisher #NW04125BOX). After electrophoresis separation at 200 V using MES running buffer (ThermoFisher #NP0002), proteins were transferred to a PVDF membrane (ThermoFisher #IB24002) using the iBlot 2 transfer stack (ThermoFisher) and the membrane was subsequently saturated with 5% milk/1xPBS for 1 h at room temperature. For detection of the protein of interest, the membrane was incubated at 4°C overnight with primary antibody (Table EV3 ) in 0.5%milk/PBS/0.05%tween and after three washes in PBS/0.05% tween, HRP‐linked secondary antibody incubation was carried on in 0.5%milk/PBS/0.05%tween for 1 h at room temperature. After washing thrice with 0.5%milk/PBS/0.05%tween, detection was performed by incubating the membrane with Pierce ECL western blot solution (ThermoFisher #32132) for 5 min prior imaging with ChemiDoc XRS+ system (BioRad).
Ib24002
Manufactured by Thermo Fisher Scientific
The IB24002 is a laboratory instrument designed for liquid handling and sample preparation. It features precise pipetting capabilities, multiple channels, and programmable automation to perform a variety of liquid handling tasks. The core function of the IB24002 is to enable accurate and efficient liquid transfer and sample processing in a laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Iblot 2 transfer stack, by Thermo Fisher Scientific (1 mentions)
B0007, by Thermo Fisher Scientific (1 mentions)
Nw04125box, by Thermo Fisher Scientific (1 mentions)
Np0002, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail set, by Merck Group (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Np0301, by Thermo Fisher Scientific (1 mentions)
Ibolt 2, by Thermo Fisher Scientific (1 mentions)
Difco 232100, by BD (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (1 mentions)
Imagej pro plus software, by Media Cybernetics (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using ib24002
1
Western Blot Protein Detection Protocol
2
Quantitative Proteomics of PAN-Interacting Proteins
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A total of 2 × 107 BCBL-1 cells were washed with 1× PBS and resuspended in nuclear lysis buffer I (10 mM HEPES pH 7.4, 20 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 1 mM TCEP, 0.5 mM phenylmethylsulphonyl fluoride [PSMF] and 0.1% NP-40) supplemented with 10 µL of murine RNase Inhibitor (200U final), and 5 µL Protease Inhibitor Cocktail Set (EMD Millipore 539134) in a total volume of 1 mL. Cells were sonicated for 180 sec at 4°C, 15 peak power, 10 duty factor, 200 cycles at 1.5 average power with the wavelength adaptor no. 500534. DNase I (NEB M0303) treatment was performed according to the manufacturer's protocol, followed by 30 min 16,000g centrifugation at 4°C. The whole-cell protein lysates and the proteins cross-linked to PAN were analyzed by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride (PDVF) membrane using a semidry blotting system (Thermo Fisher IB24002). The membranes were washed with 1× PBS with 0.1% Tween 20 (PBST) and blocked with 4% (w/v) nonfat milk in 1× PBST for 1 h at room temperature. The membranes were incubated with a specific primary antibody (Supplemental Table 2 ) for 1 h at room temperature, washed thrice with 1× PBST, and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Supplemental Table 2 ) for 45 min at room temperature. Blots were developed using the enhanced chemiluminescence substrate (Bio-Rad 1705061).
3
Optimized Immunoblotting Protocol for Nif Protein Analysis
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Immunoblotting was performed according to the method described previously (19 (link)). Briefly, samples were loaded on 10% SDS-polyacrylamide gels (Thermo Fisher Scientific; NP0301, NP0302) with 5 μL of PageRuler Prestained Protein Ladder (Thermo Fisher Scientific; 26616) as a marker. Proteins on the gels were subsequently transferred to PVDF membranes (Thermo Fisher Scientific; IB24002) using an iBolt 2 (Thermo Fisher Scientific). The membranes were blocked with 5% skim milk (BD Difco 232100; BD Biosciences) in PBS buffer and then incubated with primary antibodies for 4 to 8 h according to the sensitivity of the antibody for each Nif protein and labeled-tag. The secondary antibody goat anti-rabbit IgG-HRP (ZSGB Biotech; ZB-2301) or goat anti-mouse IgG-HRP (ZSGB Biotech; ZB-2305) was used at 1:3,000 dilution and incubated for 2 h before development. The primary antibodies used for immunoblotting and quantification in this study were as described previously (21 (link)), with the exception of different antibodies against GFP (ZSGB Biotech, TA-06), HSP60 (Proteintech, 66041-1-Ig) and Actin (HUABIO, HA601082).
4
Extracellular Matrix Protein Analysis
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Immunoblot analysis of the Collagen III, Collagen I, matrix metalloproteinase (MMP) 2 and MMP9 were performed using protein extraction from sham-operated thoracic aorta and the PTAAs (28 days after surgery). NuPAGE 4–12% Bis-Tris gel (Thermofisher, NP0335BOX), NuPAGE MES SDS running buffer (Thermofisher, NP0002) were used for protein electrophoresis. 30 μg denatured protein was added into the lane of 4–12% Bis-Tris gel (1.0–1.5 mm, 10 wells). The protein electrophoresis was performed at 110V for 1.5 h. Then, the mini-size stack (8 cm x 8 cm, Thermofisher, IB24002), an integrated pre-activated PVDF transfer membrane was used for dry protein transferring, which was finished in 7 min by using the iBlot 2 Gel Transfer Device. QuickBlock™ Blocking Buffer (Beyotime, P0252) was used for the blocking step (25°C, 1 h). Afterward, the membrane was incubated in primary antibodies with distinct dilution ratio (Supplementary Table 1 ) at 4°C for 16 h, followed by incubation with distinct secondary antibody solution (Supplementary Table 1 ) at room temperature for 1 h. Finally, images were acquired using a Tanon automatic chemiluminescence imaging system and the optical density of protein bands were determined by Image J pro plus software (Version 6.0.0.260, Media Cybernetics, Inc., Rockville, MD, USA).
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