Trans-cinnamic acid was obtained from Sigma Aldrich (C80857). Materials for primary astrocyte culture (DMEM/F-12, Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic) were obtained from Mediatech (Washington, DC). For primary neuron culture, neurobasal media, B27 supplement, B27 supplement minus antioxidant were purchased from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Atlas Biologicals. Thioflavin-S and all other molecular biology-grade chemicals were purchased from Sigma Aldrich. All primary antibodies sources and dilutions used are listed in Table S1 . Secondory antibodies (Alexa-fluor 488 and 647-conjugated) for immunostaining were purchased from Jackson ImmunoResearch. For immunoblotting, 10× Tris/Glycine/SDS running buffer was obtained from BioRad (1610772) and IR-dye-labeled secondary antibodies were obtained from Li-Cor Biosciences.
Tris glycine sds running buffer
Manufactured by Bio-Rad
Sourced in United States
Tris/Glycine/SDS running buffer is a solution used for electrophoresis in protein analysis. It is designed to maintain the pH and ionic conditions required for the separation of proteins by size through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Automatically generated - may contain errors
Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (9 mentions)
Mini protean tgx precast gel, by Bio-Rad (7 mentions)
Trans blot turbo transfer system, by Bio-Rad (7 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
Clarity western ecl substrate, by Bio-Rad (6 mentions)
Bio safe coomassie stain, by Bio-Rad (5 mentions)
Laemmli buffer, by Bio-Rad (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Criterion tgx precast gel, by Bio-Rad (4 mentions)
Trans blot turbo rta mini pvdf transfer kit, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (3 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Sodium hydroxide (naoh), by Merck Group (3 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
75 protocols using tris glycine sds running buffer
1
Astrocyte and Neuron Culture Protocols
2
Protein Visualization by SDS-PAGE
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Changes in proteins over time during incubation with pepsin, trypsin, and chymotrypsin were visualized with reducing SDS-PAGE. Laemmli sample buffer and 10× tris/glycine/SDS running buffer were obtained from Bio-Rad (Hercules, CA), β-mercaptoethanol was obtained from Sigma-Aldrich, and SeeBlue Plus2 protein standard was obtained from Invitrogen (Carlsbad, CA). Sample and sample buffer (containing 1% β-mercaptoethanol) were combined in a 1:1 ratio and the mixture was heated for 5 min at 85°C. Samples were run on a 15% tris-HCl Ready Gel (Bio-Rad Laboratories) at constant voltage (200 V). Gels were stained with Imperial Protein Stain (Thermo Fisher Scientific, Rockford, IL). Band density was quantified with UN-SCAN-IT gel analysis software (Silk Scientific, Inc., Orem, UT).
3
2D-DIGE Phosphoprotein Profiling Protocol
4
Astrocyte and Neuron Culture Protocols
5
Synthesis and Purification of Indolyl-Benzimidazoles
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SY-LB-35 and SY-LB-57 are indolyl-benzimidazoles that were synthesized and purified by HPLC by Dr. Leonard Barasa in the laboratory of Dr. Sabesan Yoganathan at St. John's University (Queens, NY, USA). Details of SY-LB synthesis, purification and characterization were previously reported [30, 31] . Cell Lysis Buffer (10×) was obtained from Cell Signaling Technologies (Danvers, MA, USA). TGX Fast Cast Acrylamide gel kit (12%), 4× Laemmli Sample Buffer, 10× Tris/Glycine/SDS Running Buffer and Precision Plus Protein Standards were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Amersham™ Protran™ 0.2 µm Nitrocellulose membranes were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Bovine Serum Albumin (30% solution) was from Alfa Aesar (Ward Hill, MA, USA). SuperSignal™ West Femto HRP Substrate solutions for chemiluminescent imaging was from Thermo Scientific (Rockford, IL, USA).
6
Protein Characterization and Quantification
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Whey protein concentrate 80 (79.6% protein) and Protease M SD (~40,000 U/g) were kindly donated by Agropur and Amano Enzyme Inc., respectively. Salibra 700 Procream (75.3% protein) was purchased from Glanbia Nutritionals, while MEG-3 30% 8A fish oil, with a minimum of 30% n-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) was purchased from DSM. Bovine trypsin (~10,000 N-α-Benzoyl-l-Arginine Ethyl Ester units/mg of protein, EC 3.4.21.4) was obtained from Millipore Sigma. Criterion TGX 8 to 16% precast gels, Precision Plus all blue molecular weight marker, Laemmli sample buffer, and 10× Tris-glycine-SDS run-ning buffer were purchased from Bio-Rad Laboratories Inc., whereas the Imperial Protein Coomassie blue stain was purchased from ThermoFisher Scientific. Analytical grade propionaldehyde and ethyl butyrate standards were purchased from Millipore Sigma. All other analytical grade reagents were purchased from Millipore Sigma or ThermoFisher.
7
Western Blot Analysis of HSP90 and LDHA
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Homogeneous tumor powder was lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount was measured with a PierceTM BCA protein Assay Kit (Thermo Scientific). Equal amounts of proteins were loaded onto 4–15% Mini-PROTEAN TGXTM Precast Gels (Bio-Rad). Following electrophoresis in 1× Tris/glycine/SDS running Buffer (Bio-Rad), proteins were transferred to PVDF membranes using the Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad) according to the vendor’s instructions. Non-specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1* Tris-Buffered Saline, 0.1% Tween 20, Bio-Rad) at room temperature for 1 h.
Membranes were incubated with primary anti-HSP90, anti-LDHA (Cell Signaling, #2021S, dilution 1:1000), in tTBS-BSA 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR) in tTBS-BSA 1% at room temperature for 1 h. Detection was performed using the SuperSignalTM West Pico Plus Kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No Background correction was performed.
Membranes were incubated with primary anti-HSP90, anti-LDHA (Cell Signaling, #2021S, dilution 1:1000), in tTBS-BSA 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR) in tTBS-BSA 1% at room temperature for 1 h. Detection was performed using the SuperSignalTM West Pico Plus Kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No Background correction was performed.
8
Optimized SDS-PAGE and Native PAGE
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Protein expressions were evaluated on 10% Mini-PROTEAN TGX Precast Protein gels from Bio-Rad. For SDS-PAGE, 3 μl of a protein solution was mixed with 4 μl of 500 mM DTT and 7 μl of the 2× SDS Sample Buffer. Then, the sample was denatured at 90 °C for 5 min, snap-cooled on ice and loaded on the gel. The general running conditions for SDS were 150 V in the 1× Tris-Glycine-SDS Running Buffer from Bio-Rad. The gels were further stained with Bio-Safe Coomassie Stain from Bio-Rad. Molecular ladders used were Broad Range Protein Molecular Weight Markers from Promega and Kaleidoscope™ Prestained Standards (#161-0324 and #161-0375) from Bio-Rad. Native PAGE was performed using the same 10% Mini-PROTEAN TGX Precast Protein gels where 12 μl of protein solution was mixed with 12 μl of 2× Native Sample Buffer from Bio-Rad prior to loading (no heat denaturation employed). For Native PAGE, the molecular ladder used was NativeMark™ Unstained Protein Standard from Thermo Fisher (#LC0725).
To analyze the proteins comprising FluoroTect GreenLys, the denaturation protocol for SDS-PAGE was changed to 70 °C for 2 min, and a protein loading per well was increased to 6 μl. After the run was complete, the fluorescent images of gels were collected immediately by FluorChem Q from Alpha Innotech or a laser-based scanner Typhoon FLA 9500 from GE Healthcare, set for Alexa488 excitation and emission.
To analyze the proteins comprising FluoroTect GreenLys, the denaturation protocol for SDS-PAGE was changed to 70 °C for 2 min, and a protein loading per well was increased to 6 μl. After the run was complete, the fluorescent images of gels were collected immediately by FluorChem Q from Alpha Innotech or a laser-based scanner Typhoon FLA 9500 from GE Healthcare, set for Alexa488 excitation and emission.
9
SDS-PAGE and Lectin Blotting Analysis
10
Western Blot Protocol for Protein Analysis
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All samples were heated for 5 min at 95 °C prior to loading. SDS-PAGE was performed by loading samples on 24-well 4 to 20% Criterion TGX Precast Gels (Bio-Rad) and running them in 1× Tris/glycine/SDS running buffer (#1610772; Bio-Rad) for 45 min at 180 V.
For Western blotting (WB), proteins were transferred onto nitrocellulose membranes (iBlot2 Gel Transfer Device with #IB23001, iBlot2 NC Regular Stacks; Invitrogen) and blocked for 1 h at RT with blocking buffer (#MB-070-010F, blocking buffer for fluorescent WB). Afterward, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 dilution in blocking buffer, seeTable 1 for antibodies). The next day, membranes were washed three times for 5 min in Tris-buffered saline + 0.1% Tween-20 detergent (TBST), followed by incubation with secondary antibodies (donkey antimouse 680 or donkey anti-rabbit 800; LI-COR Biosciences, diluted 1:10,000 in TBST) for 1 h at RT. Blots were washed again three times for 5 min in TBST and then imaged on an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometric quantification of protein bands was performed with Image Studio Lite (LI-COR Biosciences).
For Western blotting (WB), proteins were transferred onto nitrocellulose membranes (iBlot2 Gel Transfer Device with #IB23001, iBlot2 NC Regular Stacks; Invitrogen) and blocked for 1 h at RT with blocking buffer (#MB-070-010F, blocking buffer for fluorescent WB). Afterward, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 dilution in blocking buffer, see
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