Blue Native (BN)-PAGE was performed using the Novex Native-PAGE Bis-Tris gel system (Invitrogen) according to the manufacturer's directions. Briefly, total cellular proteins were extracted from mDCT15 cells using non-denaturing lysis buffer in the presence of n-dodecyl-β-d-maltoside (DDM) at a final concentration of 1%. Samples were prepared with 2.5uL of G250 Coomassie additive. Samples were electrophoresed on Native-PAGE Novex 3–12% Bis-Tris gels using ‘light blue’ cathode buffer for 1hr at 150V, followed by 1hr at 250V and transferred to PVDF membranes in NuPAGE Transfer Buffer for 2hrs at 25V. Membranes were washed using tris-buffered saline (TBS)-tween and blocked with 5% milk (Bio-Rad) in TBS-T prior to immunoblotting. Primary antibodies were allowed to incubate overnight (4°, constant rocking), with secondary incubation times for 2hr at RT.
Novex native page bis tris gel system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Novex Native PAGE Bis–Tris Gel system is a laboratory instrument designed for the separation and analysis of proteins using native polyacrylamide gel electrophoresis (Native PAGE). It is intended for use in biochemical and molecular biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Merck Group (3 mentions)
Guava easycyte 8ht flow cytometer, by Merck Group (2 mentions)
Typhoon fla 7000, by GE Healthcare (2 mentions)
Ab101465, by Abcam (1 mentions)
Hpa034780, by Abcam (1 mentions)
Bas ip tr 2025 e tritium storage phosphor screen, by GE Healthcare (1 mentions)
Wide view prestained protein size marker, by Fujifilm (1 mentions)
7 protocols using novex native page bis tris gel system
1
Native Protein Separation and Analysis
2
Iron Uptake in N. fowleri under Iron Conditions
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N. fowleri cells grown for 72 hours under iron-rich and iron-deficient conditions were washed with phosphate buffered saline (PBS) (1000 g for 15 min) and transferred to measuring buffer (50 mM glucose; 20 mM HEPES; pH 7.2). Cells were counted using a Guava easyCyte 8HT flow cytometer (Merck, Germany), and 2.5×105 cells were equally split onto a 24-well plate. To assess iron uptake, cells were supplemented with 2 μM 55Fe-citrate, 2 μM 55Fe-citrate with 1 mM ascorbate, or 6.3 μM 55Fe-transferrin. Samples were incubated at 37°C for 1 hour, and then EDTA was added to a final concentration of 1 mM to chelate extracellular iron. Cells were washed three times by measuring buffer, and the protein concentration was assessed using a BCA kit (Sigma-Aldrich, USA). Samples were diluted to equal concentrations and separated using the Novex Native PAGE Bis–Tris Gel system (4–16%; Invitrogen, USA). The gel was vacuum-dried for 2 hours and autoradiographed using a tritium storage phosphor screen. The experiment was performed in three independent replicates. If applicable, densitometry was used to compare signal strength, using Fiji distribution of ImageJ [51 (link)].
3
BN-PAGE Analysis of Mitochondrial Complexes
4
Determination of Iron-Containing Complexes
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Cells were disrupted by sonication in the presence of 0.7 % digitonin. Samples were analyzed by blue native PAGE, with the Novex Native PAGE Bis–Tris Gel System (4–16 %), according to the manufacturer’s protocol (Invitrogen). The gels were vacuum-dried and autoradiographed. More specifically (Fig. 5 ), cells were grown for 5 days in Mf medium containing different amounts of metals. The cells grown in Zn-deficient medium had previously been maintained in Zn-free medium for three months. The cells were harvested, washed once with Mf medium (without Fe, Zn and Cu) and resuspended in the same medium (at 5 x 108 cells/ml) containing 5 μM 55Fe(III)-citrate. After 3 h, the cells were harvested and whole-cell extracts were obtained and subjected to native PAGE (25 μg/lane). After autoradiography, iron-containing bands were analyzed by mass spectrometry, as previously described [68 (link)].
5
Iron Uptake in Naegleria Species
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N. fowleri and N. gruberi cells were grown in copper-rich and copper-deficient conditions for 72 h, pelleted (1,200 g, 10 min, 4°C), washed twice, resuspended in glucose-HEPES medium (50 mM glucose, 20 mM HEPES, pH 7.2) and diluted to the same cell concentration of 1 × 106 cells per sample. The cell concentration was estimated by a Guava easyCyte 8HT flow cytometer (Merck). Samples were incubated for 1 h with 1 µM 55Fe-citrate or with 1 µM 55Fe-citrate with the addition of 1 m mM ascorbate to reduce iron to the ferrous form. Uptake was stopped by the addition of 1 mM EDTA, and the cells were then washed three times with glucose-HEPES buffer, diluted to the same protein concentration [using a BCA kit (Sigma Aldrich)] and separated using the Novex Native PAGE Bis-Tris Gel system (4–16%, Invitrogen, United States). The gel was dried for 2 h in a vacuum and autoradiographed by Typhoon FLA 7000 (GE Life Sciences, United States) using a tritium storage phosphor screen (GE Life Sciences).
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N. fowleri and N. gruberi cells were grown in copper-rich and copper-deficient conditions for 72 h, pelleted (1,200 g, 10 min, 4°C), washed twice, resuspended in glucose-HEPES medium (50 mM glucose, 20 mM HEPES, pH 7.2) and diluted to the same cell concentration of 1 × 106 cells per sample. The cell concentration was estimated by a Guava easyCyte 8HT flow cytometer (Merck). Samples were incubated for 1 h with 1 µM 55Fe-citrate or with 1 µM 55Fe-citrate with the addition of 1 m mM ascorbate to reduce iron to the ferrous form. Uptake was stopped by the addition of 1 mM EDTA, and the cells were then washed three times with glucose-HEPES buffer, diluted to the same protein concentration [using a BCA kit (Sigma Aldrich)] and separated using the Novex Native PAGE Bis-Tris Gel system (4–16%, Invitrogen, United States). The gel was dried for 2 h in a vacuum and autoradiographed by Typhoon FLA 7000 (GE Life Sciences, United States) using a tritium storage phosphor screen (GE Life Sciences).
6
Radioactive Iron Uptake Assay in Giardia
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The assay was performed as recently published [88 (link)]. Briefly, trophozoites of G. intestinalis expressing Cas9 (parental cell line), ΔbolA1 and Δgrx5 were grown in TYI-S-33 culture media supplemented with 0.5μM 55Fe (29,600 MBq mg−1) in the form of ferric citrate (1:20) and incubated for 72 hours. After incubation, the cells were harvested by centrifugation and washed three times with NaCl-HEPES buffer (0.14M NaCl, 10mM HEPES, pH 7.4). Cells were disrupted by sonication in the NaCl-HEPES buffer containing 1% digitonin and cOmplete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was assessed using a BCA kit (Sigma-Aldrich), and an equal concentration of proteins was separated using Novex Native PAGE Bis–Tris Gel system (4–16%; Invitrogen) according to the manufacturer’s protocol. Gels were vacuum-dried and autoradiographed for 7 days using a BAS-IP TR 2025 E tritium storage phosphor screen (GE Healthcare) and visualized by Typhoon FLA 7000 (GE Healthcare).
7
Molecular Mass Determination of Recombinant Enzyme
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Molecular mass of the recombinant enzyme was determined by using native gradient PAGE on a Novex NativePAGE Bis-Tris gel system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12.5% polyacrylamide gel containing 0.1% SDS according to the method previously described by Laemmli [28 (link)]. Samples were boiled for 5 min in 10 mM Tris-HCl buffer (pH 6.8) containing 1% SDS and 1% 2-mercaptoethanol. A WIDE-VIEW prestained protein size marker (Fujifilm Wako, Osaka, Japan) was used as the molecular mass standard. Protein bands were visualized by staining with Rapid CBB KANTO 3S (Kanto Kagaku, Tokyo, Japan) according to the manufacturer’s instructions.
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