For immunoblot analysis, cells were rinsed twice with PBS and collected into sample buffer containing 50% Tris-Glycine SDS buffer (Novex), 45% RIPA buffer (20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet-P40, and 1% sodium deoxycholate), 5% 2-mercaptomethanol (Nacalai tesque), 1% phosphatase inhibitor (Nacalai tesque), and 1% protease inhibitor (Nacalai tesque). Subsequently, the samples were heated at 95 °C for 5 min, and each sample was loaded onto a 5–20% polyacrylamide gel (Wako). After electrophoresis and the transfer of the gels onto PVDF membranes (Merck Millipore), the membranes were fixed with 4%PFA for 30 min and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk for 30–60 min at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies for MBP (1:500, Thermo Fisher Scientific, MA1–10837), BCAS1 (1:1000, Bioss antibodies, bs-11462R), α-syn (1:1000, BD Biosciences, 610,787), PDGFRα (1:500, Santa Cruz, sc-338), or anti-β-actin (1:10000, Sigma Aldrich, A5441). This was followed by a 60 min incubation with the appropriate secondary antibodies (Novus, NB7574 and NB7160) at room temperature, with visualization by enhanced chemiluminescence (Nacalai tesque). The density of each band was quantified using Image J software.
Tris glycine sds buffer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Tris-Glycine-SDS buffer is a commonly used buffer solution in electrophoresis techniques, particularly in SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). This buffer system is designed to maintain the pH and ionic strength required for the separation and migration of proteins during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Novex wedgewell 4 20 tris glycine gel, by Thermo Fisher Scientific (4 mentions)
Immunblot pvdf membranes, by Thermo Fisher Scientific (3 mentions)
Western lightning plus ecl, by PerkinElmer (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Amersham imager 680, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Super signal west dura extended duration substrat, by Thermo Fisher Scientific (2 mentions)
Quick coomassie stain, by Serva Electrophoresis (2 mentions)
Servagel tg prime, by Serva Electrophoresis (2 mentions)
Odyssey application software 3, by LI COR (2 mentions)
Polyacrylamide gel, by Fujifilm (1 mentions)
Nb7160, by Novus Biologicals (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti mouse igg antibodies conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Tdp 43 antibody, by Proteintech (1 mentions)
Gapdh antibody, by Meridian Bioscience (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Gfp antibody, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
17 protocols using tris glycine sds buffer
1
Immunoblot Analysis for Protein Detection
2
Western Blot Protein Detection
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Samples were prepared with Tris-Glycine SDS buffer (Novex, Life Technologies) containing 10% β-ME at a 1:1 ratio (v/v) and heated at 95°C for 5 min. Five microliters of the sample was loaded to a 0.45 μm nitrocellulose blotting membrane (Cytiva-AmerstamTM ProtanTM) and incubated at 37°C for 30 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) plus 0.1% Tween 20 (TBST) for 1 h and then incubated with B cell supernatant (1:100) or mAbs (1:500) overnight at 4°C. Membranes were then washed in TBST three times for 10 min each and incubated for 1 h with donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:5000; Jackson ImmunoResearch) for 1 h at room temperature. Protein expression was visualized by enhanced chemiluminescence treatment using Western Lightning Plus-ECL (Perkin Elmer).
3
Western Blot Analysis of Protein Expression
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Samples were prepared with Tris-glycine SDS buffer (Novex, Life Technologies) containing 10% β-ME at a 1:1 ratio (v/v) and heated at 95°C for 5 min. Equal amounts of protein were loaded into 10-well 4–20% Tris-glycine gels (Novex). After transfer, blots were blocked with 5% nonfat dry milk in TBST for 1 h and then incubated with rabbit anti-sera (1:5000), B cell supernatant (1:500), mAbs (26H10, 2E9 and 23A1; 1:500), rabbit polyclonal GFP antibody (1:1000, Thermo Fisher Scientific, A-11122), rabbit polyclonal TDP-43 antibody (1:1000, Proteintech, 12892-1-AP) or mouse monoclonal GAPDH antibody (1:5000, Meridian Bioscience, H86504M) overnight at 4°C with rocking. Membranes were then washed in TBST three times for 10 min each and incubated for 1 h with donkey anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (1:5000, Jackson ImmunoResearch) for 1 h at room temperature. Protein expression was visualized by enhanced chemiluminescence treatment using Western Lightning Plus-ECL (Perkin Elmer).
4
Western Blotting of Whole Cell Lysates
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For western blotting of total lysates, cells were harvested and washed three times in 1× PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500 µM dithiothreitol) with protease inhibitors (Sigma-Aldrich). Approximately 20 μg of whole cell lysates were loaded in Novex WedgeWell 4-20% Tris-Glycine Gels (Invitrogen) in a Tris-Glycine-SDS buffer (Invitrogen) and separated by gel electrophoresis (SDS-PAGE). The proteins were then transferred to Immun-Blot PVDF membranes (Thermo Fisher Scientific) for antibody probing. Membranes were incubated with 10% bovine serum albumin in TBS with 3% Tween 20 (TBST) for 30 min at room temperature (RT), then incubated for variable times with suitable antibodies diluted in 5% bovine serum albumin in 1× TBST, washed with TBST and incubated with a dilution of 1:10,000 of the secondary antibody for 1 h at RT. The antibody was visualized using Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and imaged with Amersham Imager 680.
5
Protein Expression Analysis by Western Blot
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Total protein extracts, respectively, biotinylated protein extracts were loaded in equal amounts per lane on Novex™ WedgeWell™ 4–20% Tris‐Glycine Gel (Invitrogen, Carlsbad, USA) and after electrophoresis using Tris‐Glycine SDS buffer (invitrogen, Carlsbad, USA) transferred to PVDF membranes (Millipore, Bedford, USA). The membranes were blocked for 1 hr at room temperature in 3% skimmed milk (BIO‐RAD, Hercules, USA). The primary antibodies Zo‐1 (rabbit), (1:200, Invitrogen, Carlsbad, USA), occludin 1:200 (rabbit), (Invitrogen, Carlsbad, USA) and phospho‐caveolin‐1 (rabbit) (1:2000, abcam, Cambridge, UK) were applied to the membranes and incubated at 4°C overnight. Beta‐actin antibody was applied as loading control. After three times wash with TBS/T buffer, the secondary antibodies goat anti‐rabbit lgG‐HRP (Santa Cruz biotechnology, Santa Cruz, USA) (1:5000 diluted) and Precision ProteinTM StrepTactin‐HRP Conjugate (BIO‐RAD, Hercules, USA) (1:10000 diluted) were applied to the membranes and incubated for 1 h at room temperature. The membranes were exposed to Pierce ECL Western Blotting Substrate (Thermo, Rockford, USA) and signals detected by High performance chemi‐luminescence films (GE healthcare, little Chalfont, UK) for further analysis using Image Studio Lite (LI‐COR, Lincoln, US).
6
Protein Expression Analysis of Cell Cycle Regulators
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Whole-cell lysates were prepared in ice cold buffer containing 50 mmoL/L Tris-HCl (pH 7.4), 150 mmoL/L NaCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 1 mmoL/L Na3VO4, 20 mmoL/L NaF, 1 μg/mL aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin, and 1 μM phenylmethylsulfonyl fluoride. Protein concentrations were determined with the Bio-Rad protein assay kit using bovine serum albumin as a standard. Aliquots of 30 μg were denatured by boiling in Tris-Glycine SDS buffer (Invitrogen), separated by 12% SDS and transferred onto nitrocellulose membranes (Whatman, Dominique Dutscher, Brumath Cedex, France) by electroblotting. The membranes were blocked in 5% non-fat dry milk in 0.1% Tween 20 Tris-buffered saline for 1 h and probed with primary antibodies against cyclin E1, cyclin A2, cyclin B1, p27, p21, STAT3, p-STAT3, retinoblastoma protein (Rb), p-Rb, and actin (Santa Cruz Biotechnology).
7
Western Blot Analysis of Cell Lysates
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For total lysate, cells were harvested and washed three times in 1× PBS and lysed in radioimmunoprecipitation assay buffer (RIPA buffer) (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500 μM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4–20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS–PAGE) Tris-Glycine-SDS buffer (Invitrogen). The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing. Membranes were incubated with 10% BSA in TBST for 30 min at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1× TBST, washed with TBST, and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Full membranes (uncropped) of the blots presented in the main figures are available as source data file.
8
Whole-Cell Lysis and Western Blotting
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Whole-cell lysates were made in ice-cold lysis buffer supplemented with phosphatase and protease inhibitors (50 mM Tris-Cl pH7.4, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mmoL/L Na3VO4, 20 mmoL/L NaF, 1 μg/mL aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 1 μM phenylmethylsulfonyl fluoride). Tissue lysates were obtained from flash-frozen tissue, which was lysed by bead beating in lysis buffer. Protein quantification was performed using the Bio-Rad protein assay kit and bovine serum albumin. 30 µg of protein was resolved in a 12% polyacrylamide gel in Tris-Glycine SDS buffer (Invitrogen), before being electrotransferred onto nitrocellulose membranes (Whatman, Dominique Dutscher). Membranes were blocked in 5% non-fat milk in 0.1% Tween 20 Tris-buffered saline for 1 h and probed with the primary antibodies. AMPK (RRID:AB_915794; dilution 1:1000), Thr172P-AMPK RRID:AB_2169396; dilution 1:1000), AKT (RRID:AB_329827; dilution 1:1000), Ser473P-AKT RRID:AB_2315049; dilution 1:1000) and tubulin (RRID:AB_2288042; dilution 1:1000) antibodies were purchased from Cell Signaling Technologies. The gp130 antibody was obtained from Santa Cruz Biotechnology (RRID:AB_647629; 0.2 µg/mL).
9
Western Blot Protein Analysis Protocol
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For total lysate, cells were harvested and washed three times in 1X PBS and lysed in RIPA buffer (50mM
Tris-HCl pH7.5, 150mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500uM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4-20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS-PAGE) Tris-Glycine-SDS buffer (Invitrogen).
The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing.
Membranes were incubated with 10% BSA in TBST for 30 minutes at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1X TBST, washed with TBST and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Used antibodies are listed in the "Antibodies" section of the methods.
Tris-HCl pH7.5, 150mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 500uM DTT) with proteases inhibitors. Twenty μg of whole cell lysate were loaded in Novex WedgeWell 4-20% Tris-Glycine Gel (Invitrogen) and separated through gel electrophoresis (SDS-PAGE) Tris-Glycine-SDS buffer (Invitrogen).
The proteins were then transferred to ImmunBlot PVDF membranes (ThermoFisher) for antibody probing.
Membranes were incubated with 10% BSA in TBST for 30 minutes at room temperature (RT), then incubated for variable times with the suitable antibodies diluted in 5% BSA in 1X TBST, washed with TBST and incubated with a dilution of 1:10000 of secondary antibody for one hour at RT. The antibody was then visualized using Super Signal West Dura Extended Duration Substrat (ThermoFisher) and imaged with Amersham Imager 680. Used antibodies are listed in the "Antibodies" section of the methods.
10
Molecular Mass Analysis of Silk Fibroin
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The molecular mass distribution of silk fibroin was studied using SDS-PAGE. Silk fibroin films were dissolved in LiSCN (70%) overnight at room temperature at a concentration of 10 µg µL−1. For each sample, 30 µg was loaded on a precast vertical 4–20% Tris/Glycine gel (SERVAGel™ TG Prime™) (SERVA, Heidelberg, Germany) under reducing conditions in Tris/Glycine SDS Buffer (Fisher Scientific, Schwerte, Germany). SDS-PAGE was performed at 250 mA with a molecular mass ladder (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa) (Fisher Scientific, Schwerte, Germany); then, the silk fibroin samples were stained with a colloidal Coomassie staining solution (Quick Coomassie® Stain) (SERVA, Heidelberg, Germany).
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