Cells and tissues were washed with cold 1X PBS, harvested and lysed for 30 mins in IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol) with protease inhibitors (BioTool) and TSA (Trichostatin A, Sigma). Lysates were quantified with Bradford assay (BioRad) and immunoblotted using: SIRT3 (Cell signaling), IDH2 (Proteintech), IDH2-K413-Ac (9 (link)), actin (Sigma) and GAPDH (Millipore). For IDH2 dimerization assay, lysates were cross-linked with 0.05% glutaldehyde for 10 minutes at room temperature and immunoblotted with IDH2 antibody. For Native-PAGE analysis, flag-tagged IDH2 was transiently transfected into MCF7 or HEK-293T cells, eluted with flag-beads (Sigma), and separated on a Bis-Tris Native gel with the addition of G-250 sample additive (Invitrogen).
Bis tris native gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bis-Tris Native gel is a type of electrophoresis gel used for the separation and analysis of native (non-denatured) protein samples. It employs a Bis-Tris buffer system to maintain a neutral pH, which helps preserve the native structure and function of proteins during the separation process.
Automatically generated - may contain errors
Lab products found in correlation
Flag bead, by Merck Group (1 mentions)
Trichostatin a tsa, by Merck Group (1 mentions)
G 250 sample additive, by Thermo Fisher Scientific (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Coomassie plus protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Synergy plate reader, by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Originpro, by OriginLab (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
4 protocols using bis tris native gel
1
Analysis of IDH2 Acetylation and Dimerization
2
Blue Native-PAGE for Mitochondrial Protein Analysis
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Blue native-PAGE was performed as previously described in ref. 15 (link). The mitochondrial fraction containing 125 μg of protein was suspended in 40 μL of solubilizing buffer containing 50 μM bis-Tris (pH 7.0), 1 M aminocaproic acid, and 1.5 % DDM (n-dodecyl β-d -maltoside). Samples were cleared by centrifuging at 100,000 × g for 15 min at 4 °C. The supernatant was mixed with 3 μL of brilliant blue G (dissolved in 1 M aminocaproic acid). About 20 μL of the sample was subjected to blue native-PAGE using a 3–12% Bis-Tris native gel (Invitrogen, Carlsbad, USA). Once the dye traveled one-third of the gel length, the first cathode buffer was replaced with the second cathode buffer (10−1 dilution of the first cathode buffer).
3
Characterization of Purified YqhD Enzyme
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Integrity and purity of purified YqhD protein were checked on a native tris-bis gel (4–16%, Thermo Fisher Scientific) using 1X Native Page Running Buffer (Invitrogen). The gel was stained with Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific) for 1 h, distained overnight in water, and photographed with Bio-Rad gel doc EZ imager. The enzymatic activity of purified YqhD was determined using the method adapted from Pérez et al. (2008) (link) and measured using a Synergy plate reader at 37°C, in 200 μL of 50 mM phosphate buffer (pH 7.0) supplemented with 0.6 mM NADPH (Sigma-Aldrich), 10 μg of purified YqhD, and 0–4 mM of glutaraldehyde. The KM was calculated by OriginPro (OriginLab) using inbuilt Michaelis–Menten function.
4
Protein-DNA Binding Assay Protocol
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The purified protein was mixed with probe and binding buffer and incubated for 45 min in ice. The binding buffer consisted of 50 mM NaCl, 20 mM TrisCl, 1 mM DTT, and 10% glycerol. The probe was prepared previously by amplifying genomic DNA using primers 19 and 20 (Table 2 ) and gel purification. A negative control probe (non-binding DNA) was prepared from the amplification of DNA, and its sequence is shown in Table 2 , item 27. After incubation, the mixture was run in native tris-bis gel (4–16%, Thermo Fisher Scientific) with TBE as running buffer for about 2 h at 4°C and 100–150 V. The gel was stained with SyberGreen according to the Electrophoretic Mobility Shift Assay (EMSA) Kit E33075 (Invitrogen) and photographed with Biorad gel doc EZ imager.
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