Precast NuPAGE 4-12% Bis-Tris polyacrylamide protein gels were purchased from Invitrogen. Protein gels were run at 200 V for 35 minutes in MES buffer, gels were then washed 9 times with warm water, stained with coomassie blue for 4 hours. It was then destained using tap water for 16 hours before we imaged them on Bio-Rad ChemiDoc MP imaging System. All protein bands were analyzed and quantified using Imagelab 5.2.1. Agarose DNA gels were made in house using 1% UltraPure Agarose 1000 in TAE buffer with 0.01% EZ-Vision (Amresco), DNA gels were run at 150 V for 25 minutes and visualized under UV.
Nupage 4 12 bis tris protein polyacrylamide gels
Manufactured by Thermo Fisher Scientific
The NuPAGE 4–12% Bis-Tris Protein polyacrylamide gels are precast gel electrophoresis products designed for the separation and analysis of proteins. They feature a bis-tris buffer system and a gradient of 4% to 12% polyacrylamide concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Ez vision, by Avantor (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Westernbright quantum, by Advansta (1 mentions)
5 protocols using nupage 4 12 bis tris protein polyacrylamide gels
1
Protein and DNA Gel Electrophoresis
2
Protein and DNA Gel Electrophoresis
3
Western Blot Analysis of TRKB Signaling
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The levels of pTRKB and total TRKB in the prefrontal cortex of experimentally naive adult male mice were determined by WB as previously described (Rantamäki et al. 2007 (link)). Samples were homogenized as mentioned above and denatured in a 2X Laemmli buffer (BioRad, Cat# 1610737) for 5 min at 95 ℃. SDS-PAGE was carried out by resolving the samples by electrophoresis in NuPAGE 4–12% Bis–Tris Protein polyacrylamide gels (Cat# NP0323BOX, Invitrogen). After the electrophoresis, the samples were transferred to a PVDF membrane and incubated in primary antibody diluted 1:1000 in 3% BSA/TBST overnight at 4 ℃. The membrane was subsequently washed and incubated in HRP-conjugated secondary antibody against the appropriate host (1:10,000, BioRad) for 1 h of RT. The bands were visualized using Pierce™ ECL Plus western blotting substrate (#32132 Thermo-Fisher Scientific). Primary antibodies used were rabbit anti-phosphorylated TRKB against Y515, Y706, or Y816 (Cell Signaling #4619, #4621, and #4168, respectively); goat anti-TRKB (R&D System, #AF1494) or anti-β-actin mouse monoclonal antibody (Sigma-Aldrich, #A1978). Considering that HEK293T cells virtually do not express TRKB, all the antibodies against TRKB were validated by comparing non-transfected cells with TRKB-transfected cells (data not shown). To ensure the quality of the signal, the membranes were stripped only once.
4
TRKB Signaling Pathway Immunoprecipitation and Western Blot
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The cells were lysed using NP lysis buffer containing 2 mm sodium orthovanadate and protease inhibitor mix. The homogenized suspension was centrifuged (15,000 × g, 10 min, +4°C), and the resulting supernatant was used for analysis. For immunoprecipitation, TRKB was captured using anti-TRKB antibodies (R&D Systems). The samples were incubated with Sepharose, washed with NP lysis buffer twice and the proteins were separated by heating in 2× Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, and 125 mm Tris HCl; pH 6.8) for 5 min at 95°C. The samples were loaded to NuPAGE 4–12% Bis-Tris Protein polyacrylamide gels (Invitrogen, #NP0323BOX), and the proteins were separated according to their molecular weight using electrophoresis. The samples were transferred to polyvinylidene difluoride (PVDF) membrane, incubated in 1:1000 primary antibody dilution in 3% bovine serum albumin (BSA) in tris-phosphate buffer containing 0.1% Tween 20 (TBST) overnight at 4°C and subsequently incubated in HRP-conjugated secondary antibodies (1:10,000) for 1 h at room temperature (RT). The bands were visualized using chemiluminescent western blotting substrate in Fuji LAS3000 camera (Tamro Medlabs).
5
Western Blot Analysis of Mouse Proteins
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Samples from male and female mice (2 months old) were used for this experiment. Forty microgram of total proteins from each sample was heated in 2X Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue and 125 mM Tris HCl, pH 6.8) for 5 min at 95°C and loaded to NuPAGE 4–12% Bis-Tris Protein polyacrylamide gels (Invitrogen, #NP0323BOX). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, blocked in the blocking buffer for 1 h (3% BSA in Tris-PB: TBST, 20 mM Tris-HCl; 150 mM NaCl; 0.1% Tween-20; pH 7.6) and incubated with the primary antibody diluted 1:1,000 in the blocking buffer overnight at 4°C. The membranes were subsequently incubated in HRP-conjugated secondary antibodies diluted in the blocking buffer (1:10,000) for 1 h at room temperature. The signal was detected using enhanced chemiluminescent (ECL) HRP substrate WesternBright Quantum (Advansta, #K-12042) in a CCD camera (G:Box Chemi, Syngene). The levels of phospho-proteins analyzed by Western blot were normalized by total levels of the corresponding proteins; the levels of total TRKB, PSD-93, and PSD-95 were normalized by B-actin. PSD-95 samples from PFC were not normalized due to low detection signal from the housekeeping protein.
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