For every experiment using [γ−32P]ATP, we used a 1:20 mix of 10 μCi (6,000 Ci/mmol; PerkinElmer) and unlabeled ATP (100 μM unless otherwise noted). All radioactive protein samples were separated by size on a 4-to-20% Mini-Protean TGX precast SDS-PAGE gel (Bio-Rad). The gel was then dried and imaged using a Typhoon phosphorimager (Molecular Dynamics GE Healthcare Life Sciences). All biochemical experiments were conducted in at least triplicate unless otherwise noted.
Mini protean tgx precast sds page gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN TGX Precast SDS-PAGE gels are polyacrylamide gels pre-cast for use in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations. They are designed for rapid and consistent protein separation and analysis.
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Lab products found in correlation
Illustra gfx pcr dna and gel band purification kit, by GE Healthcare (2 mentions)
Hispur nickel nitrilotriacetic acid agarose ni nta resin, by Thermo Fisher Scientific (2 mentions)
Pierce protease inhibitor tablet, by Thermo Fisher Scientific (2 mentions)
Du 730 life sciences uv vis spectrophotometer, by Beckman Coulter (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Gelcode, by Thermo Fisher Scientific (1 mentions)
Acss2, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ap132p, by Merck Group (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Acss2, by Thermo Fisher Scientific (1 mentions)
Total acetylated lysine, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Trans blot turbo apparatus, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
11 protocols using mini protean tgx precast sds page gel
1
Radioactive Protein Analysis Protocol
2
Proteinase K-Treated Lactoferrin Analysis
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LF was treated with 100 µg/mL proteinase K for 1 h at 46 °C followed by 10 min on 95 °C to inactivate the enzyme activity. 1 µg of LF and proteinase K treated LF were loaded on a SDS-page gel (Mini-PROTEAN TGX Precast SDS-page gel, Biorad, Berkeley, CA, USA) and run on 120 V for 1 h. The gel was stained with GelCode (Thermo Scientific, 24590, Waltham, MA, USA) according to the manufacturer’s instructions.
3
Acetylome Analysis of ACLY and ACSS2 KOs
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A549, HepG2, HAP1, and 634T WT, ACLY and/or ACSS2-KO cell lines were lysed in mammalian protein extraction reagent (M-PER) buffer (Thermo Fisher Scientific, 78501) with 1× protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225). Fifteen micrograms of protein was loaded and separated using 4 to 15% Mini-PROTEAN TGX Precast SDS-PAGE Gels (Bio-Rad, #4561086). Samples were then transferred to nitrocellulose membranes for immunoblotting. Total acetylated-lysine (Cell Signaling Technology, 9441), β-actin (Sigma-Aldrich, A5441), ACLY (Cell Signaling Technology, 13390), ACSS2 (Cell Signaling Technology, 3658), and PEX5(D7V4D) (Cell Signaling Technology, 83020) antibodies were used to probe their respective targets. Anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Millipore, AP132P) were used for imaging.
4
MERS PLpro Reactivity Assays
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Probe reactivity assays were set-up at a 10 μL scale with pure MERS PLpro enzymes and PA probes. The reactivity of MERS PLpro wild-type was assessed with both PA probes at varying molar ratios. Ub-PA and human ISG15-PA were diluted in storage buffer containing fresh 10 mM DTT at final concentrations ranging from 5 to 25 μM and 25–100 μM, respectively. Each reaction was initiated with 5 μM MERS PLpro wild-type and allowed to react for 1 h at room temperature before quenching with 5X loading buffer (250 mM Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 0.02% bromophenyl blue, and fresh 400 mM DTT). The reactivity of MERS PLpro wild-type and mutants were evaluated at single molar ratios of 5 μM PLpro and either 25 μM Ub-PA (1:5 M ratio) and 100 μM ISG15-PA (1:20 M ratio), which were quenched at different time points (2, 5, 30 min) with loading buffer. The entire reaction was loaded onto 4–20% Mini-PROTEAN® TGX™ precast SDS-PAGE gels from BioRad, and proteins were visualized with Coomassie Brilliant Blue staining.
5
Protein Purification and Analysis
6
Western Blot Analysis of IFITM Proteins
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Total protein was quantified by a bicinchoninic acid (BCA) assay (Thermo Scientific), and equal amounts of protein were loaded onto Mini-Protean TGX precast SDS-PAGE gels (Bio-Rad). Proteins were transferred onto nitrocellulose membranes using a TransBlot Turbo apparatus (Bio-Rad). Nitrocellulose membranes were blocked overnight using 5% (wt/vol) milk powder–PBS-Tween. Proteins were visualized with the following primary antibodies: human IFITM3 (rabbit anti-IFITM3, N-terminal amino acids 8 to 38, catalog number AP1153a; Abgent), human IFITM1 (rabbit anti-IFITM1, catalog number HPA004810; Sigma), and β-actin (rabbit anti-β actin, catalog number ab8227; Abcam), which was used as a loading control. All primary antibodies were visualized using species-specific horseradish peroxidase-conjugated secondary antibodies (Dako).
7
Protein Purification and Analysis
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Oligonucleotide primers were synthesized by Integrated DNA Technologies or Sigma-Aldrich. Recombinant plasmid DNA was purified with a QIAprep® Spin Miniprep Kit from Qiagen. Gel extraction of DNA fragments and restriction endonuclease clean up were performed using an Illustra GFX PCR DNA and Gel Band Purification Kit from GE Healthcare. DNA sequencing was performed by Beckman Coulter Genomics and Eton Bioscience. Optical densities of E. coli cultures were determined with a DU 730 Life Sciences UV/Vis spectrophotometer (Beckman Coulter) by measuring absorbance at 600 nm. All chemicals and solvents were obtained from Sigma-Aldrich except where noted.
During protein purification, proteolysis was inhibited by adding Pierce™ Protease Inhibitor tablets (EDTA free; Thermo Scientific) to lysis buffer. Affinity chromatography and SDS-PAGE analysis were conducted using HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Thermo) and 10% or 4–15% Mini-PROTEAN TGX Precast SDS-PAGE gels (Bio-Rad) with 2× Laemmli sample buffer (Bio-Rad). Protein concentrations were determined by measuring absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo).
During protein purification, proteolysis was inhibited by adding Pierce™ Protease Inhibitor tablets (EDTA free; Thermo Scientific) to lysis buffer. Affinity chromatography and SDS-PAGE analysis were conducted using HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Thermo) and 10% or 4–15% Mini-PROTEAN TGX Precast SDS-PAGE gels (Bio-Rad) with 2× Laemmli sample buffer (Bio-Rad). Protein concentrations were determined by measuring absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo).
8
Western Blot Analysis of Leukocyte and EV Proteins
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Isolated leukocytes and EVs were solubilized in 8 M urea containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentration was assayed by the Micro BCA protein assay kit (ThermoFisher Scientific, Grand Island, NY). Lysates containing 10–30 μg leukocyte protein, or 10 μg of EV protein, were separated by electrophoresis on 4–20% Mini-PROTEAN® TGX™ Precast SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) (ThermoFisher Scientific, Grand Island, NY) for 1 h at room temperature, followed by incubation overnight at 4 °C with the CLN-5 antibody (1:200; Life Technologies, Carlsbad, CA) diluted in 5% BSA in TBST. Following incubation with anti-mouse HRP-conjugated secondary antibody (1:400; Cell Signaling), blots were developed using the chemiluminescent HRP substrate kit (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, Grand Island, NY) and signal detected using a G:Box XX6 digital gel imager (Syngene, Frederick, MD). Images were acquired by GeneSys software (Syngene, Frederick, MD).
9
Quantifying GFP-Lifeact Expression
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For probing the expression level of GFP-Lifeact, exponentially growing cells (50 ml) were harvested by centrifugation at 3000 rpm for 5 min. The pellet was resuspended in 300 µl of lysis buffer (50 mM Tris-HCl, 100 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% Triton X-100) containing Halt protease inhibitor cocktail (100×; #1862209; Thermo-Fisher Scientific) and frozen at –20°C. The cells were mechanically lysed using a BeadBug microtube homogenizer (Benchmark Scientific). Protein samples were resolved by Mini-PROTEAN TGX Precast SDS–PAGE gels (#4561084; BioRad). The gels were either stained with Coomassie blue or transferred to PVDF (polyvinylidene fluoride) membrane (Millipore) for immunoblots. The blots were first incubated with the anti-GFP antibody (1:1000 diluted; #11814460001; Sigma-Roche) overnight at 4°C, followed by horseradish peroxidase–linked secondary antibody (1:5000; #sc-516102; Santa Cruz) for 2 h at room temperature. Blots were developed with SuperSignal West Pico PLUS chemiluminescent substrate (#34577; Thermo-Fisher Scientific).
10
GLUT-1 and Nb-GLUT-1 Purification and Detection
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Full-length GLUT-1 and Nb-GLUT-1 were purified according to the protocol above and the concentrations were determined by comparing with the albumin standards (Thermo Scientific) on Coomassie-stained gels. Purified GLUT-1 and Nb-GLUT-1 samples were run on 12% or 4-20% gradient Mini PROTEAN TGX pre-cast SDS-PAGE gels (Bio-Rad Laboratories, Inc.). The samples on the gels were transferred on to nitrocellulose/PVDF membranes with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). Blocking was performed with either 5% skimmed milk in TBST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20; up to 0.1% Tween-20 was used at times to reduce background noise) for 30 min or PVA (polyvinyl alcohol) diluted 1:50 in TBST for 2 min. Washes and antibody treatments were conducted either traditionally or with the SNAPid system (EMD Millipore Corporation). For detecting GLUT-1, purified Nb-GLUT-1 at 2 mg/mL was used as the primary antibody at 1:1000 dilution in TBST, and HRP-rProtein A (Life Technologies) as the secondary antibody at 1:5000 dilution in TBST. For detecting Nb-GLUT-1, 1:5000 HRP-rProtein A was used directly without requiring a secondary antibody. SuperSignal West Femto Chemiluminescent Substrate was used to develop the blots (Thermo Scientific).
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