BN-PAGE and CBB-free native PAGE analyses were performed using a NativePAGETM Novex Bis-Tris Gel System (Invitrogen) according to the manufacturer’s protocols with some modifications. In brief, proteins were incubated in buffer C at 30 °C, and final concentrations of KaiA, KaiB and KaiC were 2.4, 7.0 and 7.0 μM, respectively. Aliquots of incubated proteins were suspended in sample buffer immediately prior to electrophoresis and incubated reaction mixtures were not frozen before electrophoresis. To prepare samples for BN-PAGE, 12 μl reaction mixtures were mixed with 8 μl of sample buffer containing 1× Native PAGE sample buffer (Invitrogen), 0.25% CBB-G250, 1 mM ATP and 5 mM MgCl2 (final concentrations). Samples were then loaded onto NativePAGETM 4–16% Bis-Tris Protein Gels (Invitrogen) and were electrophoresed. For both of the anode- and cathode-sides buffers, Native PAGE Running buffer (Invitrogen) was modified to contain 5 mM MgCl2 and 0.25 mM EDTA. In addition, 0.02% CBB, 1 mM ATP were added to the cathode-side buffer. The NativeMark protein standard (Invitrogen) was used as a molecular weight marker. For CBB-free native PAGE analyses, aliquots of incubated reaction mixtures in the sample buffer (1× Native PAGE sample buffer; Invitrogen) containing 1 mM ATP and 5 mM MgCl2 were electrophoresed in a buffer containing 192 mM Glycine, 25 mM Tris and 1 mM ATP.
Nativepage sample buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
NativePAGE sample buffer is a product designed for use in Native PAGE (Polyacrylamide Gel Electrophoresis) techniques. It is used to prepare and load protein samples for electrophoretic separation under non-denaturing conditions, preserving the native structure and interactions of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nativemark unstained protein standard, by Thermo Fisher Scientific (17 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (15 mentions)
Nativepage bis tris gel, by Thermo Fisher Scientific (11 mentions)
Digitonin, by Thermo Fisher Scientific (8 mentions)
Nativepage 5 g 250 sample additive, by Thermo Fisher Scientific (8 mentions)
Digitonin, by Merck Group (6 mentions)
Nativepage gel, by Thermo Fisher Scientific (5 mentions)
Nativepage novex bis tris gel system, by Thermo Fisher Scientific (5 mentions)
G 250 sample additive, by Thermo Fisher Scientific (5 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (4 mentions)
Nativepage 4 12 bis tris gel, by Thermo Fisher Scientific (4 mentions)
Nativepage novex bis tris gel, by Thermo Fisher Scientific (4 mentions)
Nativepage system, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (3 mentions)
Dark blue cathode buffer, by Thermo Fisher Scientific (3 mentions)
Nativepage 4 16 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nativepage cathode buffer additive, by Thermo Fisher Scientific (3 mentions)
Native precast gel, by Thermo Fisher Scientific (2 mentions)
Comassie g 250, by Thermo Fisher Scientific (2 mentions)
108 protocols using nativepage sample buffer
1
Native PAGE Analysis of Circadian Clock Proteins
2
Blue-native PAGE Analysis of Protein Complexes
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Blue-native PAGE was performed using the NativePAGE Novex Bis-Tris gel system (Invitrogen) according to the instructions provided by the manufacturer. Cells were trypsinized and washed in PBS, after which the cell pellet was resuspended in lysis buffer consisting of either 1× NativePAGE sample buffer (Invitrogen) supplemented with 1% Triton X-100 or 1× NativePAGE sample buffer supplemented with 1% Triton X-100 and 4 M urea. When indicated, SDS was added to the samples to a final concentration of 1%. Alternatively, cells were lysed in radioimmunoprecipitation buffer consisting of 10 mM sodium phosphate, pH 7, 150 mM NaCl, 1% Nonidet P40, 1% deoxycholate, 0.1% SDS, 2 mM EDTA, 50 mM NaF, 100 μM sodium vanadate, and protease inhibitor cocktail (Sigma-Aldrich).
3
Native Blue Native PAGE for Mitochondrial Complexes
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BN-PAGE was performed using NativePAGE gels from Life Technologies, following the manufacturer’s instructions. Mitochondria were suspended in native PAGE sample buffer (Life Technologies) supplemented with digitonin and protease inhibitors and incubated on ice for 20 min. The digitonin:protein ratio used was 10:3. Following centrifugation at 20,000 g for 30 min, the supernatant was recovered, mixed with the G-250 sample additive (Life Technologies) and native PAGE sample buffer (Life Technologies), and loaded onto 3–12% precast Bis–Tris Native PAGE gels (Life Technologies). The NativeMark Protein standard (Life Technologies), run together with the samples, was used to estimate the molecular weight of the protein complexes. Electrophoreses was performed using the Native PAGE Running buffer (as anode buffer; Life Technologies) and the Native PAGE Running buffer containing 0.4% Coomassie G-250 (cathode buffer). Gels were stained with the Novex Colloidal Blue staining kit (Life Technologies) to reveal the protein complexes.
4
Mitochondrial Complex I Activity Assay
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Assays were performed as in ref. 71 (link). Mitochondria were purified from ten thoraces of 7–10-day-old male flies and BN-PAGE was performed using NativePAGE gels from Life Technologies, following the manufacturer’s instructions. Mitochondria were suspended in NativePAGE sample buffer (Life Technologies) supplemented with 1% digitonin and protease inhibitors, incubated on ice for 20 min and centrifuged at 20,000 × g for 30 min at 4 °C, the supernatant was recovered, G-250 (Life Technologies) sample additive and NativePAGE sample buffer was added before loading onto 3–12% precast Bis–Tris NativePAGE gels (Life Technologies). Electrophoreses was performed using the NativePAGE Running buffer (as anode buffer, from Life technologies) and the NativePAGE Running buffer containing 0.4% Coomassie G-250 (cathode buffer). Protein complexes were revealed by staining with Novex Colloidal Blue staining kit (Life Technologies). Complex I activity in native gels was performed by incubating gels in 0.1 mg/mL NADH, 2.5 mg/mL nitrotetrazolium blue chloride, 5 mM Tris-HCl (pH 7.4) overnight at room temperature. Gels were imaged using a BioRad station and densitometry was performed in ImageJ.
5
Native PAGE Analysis of OXPHOS Complexes
6
Native-PAGE Immunoblotting of Mitochondrial Complexes
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The indicated purified mitochondrial pellet was lysed in 1× NativePAGE sample buffer (BN2003, Invitrogen) supplemented with 1% (w/v) Digitonin for 30 min on ice, and 30 μg of cleared lysates was mixed with G-250 (BN2004, Invitrogen) to a final concentration of 0.25%. Samples were then loaded on 4 to 16% bis-tris NativePAGE (BN1002BOX, Invitrogen) following the manufacturer’s instructions, transferred on nitrocellulose membrane, and subjected to immunoblotting with the indicated antibodies.
7
Mitochondrial Protein Complex Profiling
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Pellets of mitochondria isolated from adult flies of the indicated genotypes were suspended at 10 mg/ml in 1× native PAGE sample buffer (Invitrogen) supplemented with protease inhibitor mixture (Sigma) and 2% (w/v) digitonin (Invitrogen). Samples were then immediately centrifuged at 100,000 g for 25 min at 4°C. The supernatant was transferred to a new tube, and the native PAGE 5% G‐250 sample additive (Invitrogen) was added. Samples were quickly loaded into a blue native polyacrylamide 3%–12% gradient gel (Invitrogen). After this step, electrophoresis was run in cathode buffer in darkness for 20 min at 150 V, and the condition was then changed to cathode buffer in the light and run for approximately 2 h at 250 V and 4°C. After electrophoresis, gels were fixed in 50% methanol +10% acetic acid for 20 min at room temperature, stained using a Colloidal Blue staining kit (Invitrogen) overnight at room temperature, and destained with deionized water.
8
Isolation and Analysis of Mitochondrial Complexes
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Approximately 50 mg of muscle tissue was minced and placed in 1 mL mitochondria isolation buffer (MIB: 0.28 M sucrose, 10 mM HEPES, pH 7.4, 2 mM EDTA) supplemented with protease and phosphatase inhibitors. Tissue was homogenized in MIB with a Dounce homogenizer, using 10 strokes each of loose and tight pestles. Homogenates were subjected to a 5 min, 2000 g spin to pellet nuclei and the resulting supernatant was subjected to 13,000 g spin followed by two washes in 1 mL MIB. Mitochondrial pellets were suspended in 100 µL MIB and subjected to bicinchoninic acid, (BCA) protein assay (Pierce). Equal amounts of mitochondrial protein ∼30 µg were solubilized in 60 µL 1× native-PAGE sample buffer (Invitrogen) supplemented with 1% Digitonin (Invitrogen). Following solubilization, a second BCA assay was performed and equal amounts of protein (∼10 µg) in 1× sample buffer with 0.25% Coomassie G250 were loaded on Invitrogen 4–16% NativePAGE gels. Gels were run for 1 h at 150 V followed by 250 V for 2.5 h. Gels were blotted to nitrocellulose membranes and probed for complex IV (Cox IV; Abcam), complex I (Ndufa9; Abcam), and complex III (UQCRFS1; Abcam), in that order with stripping in between with standard SDS/mercaptoethanol stripping buffer.
9
Native PAGE Protein Analysis
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Purified proteins were mixed with NativePAGE sample buffer (ThermoFisher) and loaded into a NativePAGE 4–12% Bis-Tris gel (Thermo Fisher) according to the manufacturer’s instruction. The BN-PAGE gels were run for 2h at 150V and stained with Coomassie blue.
10
Western Blot Analysis of C-Reactive Protein
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Total proteins (20 µg) were denatured at 70°C for 10 min, mixed with NativePAGE Sample Buffer (Thermo Fisher Scientific, Inc.), electrophoresed on Novex 4–12% Tris Glycine Midi Protein Gels (Thermo Fisher Scientific, Inc.) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% milk for 1 h and incubated with anti-human CRP antibodies overnight: Mouse anti-CRP (1:500; clone, C6; catalog no. M86284M; Meridian Life Science, Inc., Cincinnati, OH, USA), Mouse anti-CRP (1:500; clone, C5; catalog no. M86005M; Meridian Life Science, Inc.) and Mouse anti-CRP (1:500; polyclonal; catalog no. ab52687; Abcam, Cambridge, MA, USA). The membranes were washed 3 times with TBST (1% serum albumin in 50 mM Tris-HCl, pH 7.4, containing 0.05% Tween-20) at room temperature and incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2,000; catalog no. sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Membranes were washed three times, visualized with SuperSignal West Pico Chemilluminescent substrate (Thermo Fisher Scientific, Inc.) for 5 min and exposed to X-ray film. Densitometry was performed using Image J 2.1.4.7 software (National Institutes of Health).
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