Mini protean tgx precast polyacrylamide gel

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The Mini-PROTEAN TGX precast polyacrylamide gels are a laboratory equipment product manufactured by Bio-Rad. They are designed for performing electrophoresis to separate biomolecules such as proteins or nucleic acids based on their molecular weight or size.

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Trans blot turbo transfer system, by Bio-Rad (6 mentions) Nitrocellulose membrane, by Bio-Rad (4 mentions) Pvdf membrane, by Merck Group (3 mentions) Image lab software, by Bio-Rad (3 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Hybond, by Cytiva (2 mentions) X omat, by Kodak (2 mentions) Ecl western blotting system, by GE Healthcare (2 mentions) Donkey anti rabbit or mouse hrp, by Jackson ImmunoResearch (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions) Chemicapt 3000 imaging system, by Vilber (2 mentions) Supersignal west pico, by Thermo Fisher Scientific (2 mentions) Protease and phosphatase inhibitor cocktail, by Roche (2 mentions) Versadoc imaging station, by Bio-Rad (2 mentions) Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Molecular imager chemidoc xrs, by Bio-Rad (2 mentions) A00915, by GenScript (2 mentions) Ab18256, by Abcam (2 mentions)

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46 protocols using mini protean tgx precast polyacrylamide gel

Isolation and Analysis of Rat Brain Synaptic Proteins

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Brains from adult Sprague Dawley rats were supplied by Rockland Immunochemicals Inc. (Gilbertsville, PA). Animals were subjected to 1 min CO₂ prior to decapitation. Brains were collected within two minutes of decapitation and were immediately frozen in liquid nitrogen. Brains were rapidly thawed in isotonic sucrose solution and dissected to remove white matter.
Cerebral cortices were homogenized in isotonic sucrose. Synaptosome and PSD fractions were prepared essentially as described previously [21 (link)]. Proteins in homogenate, synaptosome, and PSD fractions were resolved by SDS-PAGE using 4–15% Mini-PROTEAN TGX Precast polyacrylamide gels (BioRad). Gels were transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (BioRad), blocked, incubated with primary and secondary antibodies, and visualized via chemiluminescence (BioRad).

Dosemeci A., Burch A., Loo H., Toy D, & Tao-Cheng J.H. (2017). IRSp53 accumulates at the postsynaptic density under excitatory conditions. PLoS ONE, 12(12), e0190250.

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Western Blot Analysis of Cellular Proteins

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The cells were lysed in RIPA Lysis Buffer containing Protease Inhibitor Cocktail and the phosphatase inhibitor PhosSTOP (both from Roche Applied Science, Basel, Switzerland). The protein concentration of the whole cell lysate was determined by the BCA assay (Beyotime Biotechnology, Haimen, Jiangsu, China). Proteins from the cell lysates were loaded onto 4–15% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA). Then, the proteins were transferred onto PVDF membranes (Bio-Rad Laboratories). After blocking, the membranes were sequentially incubated with the indicated primary and secondary antibodies (Cell Signaling Technology). The protein bands were visualized using an Immun-Star WesternC Chemiluminescence Kit (170–5070, Bio-Rad Laboratories) using a chemiluminescence imaging method. The band intensities were quantified using Quantity One. β-Actin or GAPDH severed as the loading control. LC3 Control Cell Extracts (#11972) and p70 S6 Kinase Control Cell Extracts (#9203) (Cell Signaling Technology) were used to identify the sensitivity of antibodies, including anti-LC3, anti-phosphorylated mTOR and anti-substrates of mTOR (Fig. S1a,b).

Chen X., Li M., Li L., Xu S., Huang D., Ju M., Huang J., Chen K, & Gu H. (2016). Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway. Scientific Reports, 6, 28423.

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Immunoblotting of Androgen Receptor and FKBP5

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Cells were seeded at 2 x 10 5 cells per well in 6-well plates in stripped medium and incubated for 72 h. Cells were treated with either V, or 10 nmol/L DHT for 16 h. Whole-cell lysates were prepared and 15 g of protein was resolved by denaturing electrophoresis on 4-15% Mini-Protean TGX precast polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA), transferred to Hybond-C membrane (Amersham Biosciences, Castle Hill, NSW, Australia), and immnostained using 1:10,000 rabbit anti-human AR (N-20) polyclonal IgG, 1:4000 rabbit anti-human FKBP5 (H-100) polyclonal IgG, and 1:5000 mouse anti-human -actin (clone AC-15) polyclonal IgG1 (Sigma-Aldrich, St Louis, MO). Immunoreactivity was detected using the appropriate horseradish peroxidase-conjugated IgG and visualized using enhanced chemiluminescence (Amersham).

Smith E., Palethorpe H.M., Ruszkiewicz A.R., Edwards S., Leach D.A., Underwood T.J., Need E.F, & Drew P.A. (2016). Androgen Receptor and Androgen-Responsive Gene FKBP5 Are Independent Prognostic Indicators for Esophageal Adenocarcinoma. Digestive diseases and sciences, 61(2).

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Western Blot Analysis of Stress Proteins

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Lyophilised pellets were Dounce-homogenized on ice in about 250 lL of lysis buffer [20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton], supplemented with a protease inhibitor cocktail (Roche) and phoshatase inhibitor cocktail (SIGMA). Total cell lysate (30 lg) per each cell extract was run on a 4-15% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Amersham UK). After blocking, replicate membranes were incubated overnight at 4 C with either one of the following primary antibodies: (i) Heat Shock Protein 70 (SIGMA, Cat N. H-5147) 1:1000; (ii) Heat Shock Protein 60 (SIGMA, Cat N. H-3524) 1:250; (iii) Phospho-p38 MAP Kinase (Thr180/Tyr182) (Cell Signalling, 9211) 1:250; (iv) Manganese superoxide dismutase (Enzo Life Sciences, ADI-SOD-110) 1:500. Membranes were washed three times with PBS-Tween 20 prior to incubation with a fluorescein-labelled secondary antimouse and/or -rabbit antibody (LI-COR Biosciences). Protein levels (Hsp70, Hsp60, MnSOD, P-p38 Mapk) were normalised using a-tubulin as internal control. Results were reported in arbitrary units ± SE obtained from the volumetric analysis of the normalised bands (sum of the two independent experiments).

Pinsino A., Bergami E., Della Torre C., Vannuccini M.L., Addis P., Secci M., Dawson K.A., Matranga V, & Corsi I. (2017). Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos. Nanotoxicology, 11(2).

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Western Blot Analysis of Exosomal Proteins

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Approximately, 5–10 μg of protein were run on 4–20% Mini-PROTEAN®TGX™ precast polyacrylamide gels (Bio-Rad Laboratories, Inc., Hercules, CA), transferred to nitrocellulose membranes, blocked, and probed with primary antibodies for: CD63, ALIX, TSG101, Actin, CK7, CK19, GGT1 diluted 1:500 (Santa Cruz Biotechnology). The secondary antibody, donkey-anti-rabbit or mouse-HRP (Jackson Labs, West Grove, PA) was diluted 1:10,000. The signal was developed with the ECL Western Blotting System (GE Healthcare, Little Chalfont UK) using a Kodak X-Omat (Rochester, NY).

Loarca L., De Assuncao T.M., Jalan-Sakrikar N., Bronk S., Krishan A., Huang B., Morton L., Trussoni C., Bonilla L.M., Krueger E., O’Hara S., Splinter P., Shi G., Pisarello M.J., Gores G.J., Huebert R.C, & LaRusso N.F. (2017). Development and Characterization of Cholangioids from Normal and Diseased Human Cholangiocytes as an In Vitro Model to Study Primary Sclerosing Cholangitis. Laboratory investigation; a journal of technical methods and pathology, 97(11), 1385-1396.

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Phospho-Akt Protein Extraction and Detection

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Total proteins were isolated from GBM cells by sonication in a lysis buffer composed of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 2.5 mM EGTA, pH 7.4, 0.1% Tween 20, 10% glycerol, 0.1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 mM glycerophosphate and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Proteins (20 μg) were resolved on 4-20% Mini-PROTEAN® TGX™ precast polyacrylamide gels (Bio-Rad, Marnes-la-Coquette, France) and transferred to an Amersham GE Healthcare PVDF membrane (0.45 μm pore size) (Fisher Scientific, Illkirch, France). The following antibodies were used: a rabbit anti-human Phospho-Akt (Ser473) (#4058, clone 193H12, Ozyme, St Quentin en Yvelines, France), rabbit anti-human Akt (#9272, Ozyme). They were diluted at a ratio of 1:1000 according to the manufacturer's instructions. Goat anti-Rabbit and anti-Mouse IgG Secondary Antibody, HRP conjugate (Fisher Scientific, Illkirch, France) was used at a dilution of 1:2000. Detection was performed on SuperSignal™ West Femto Maximum Sensitivity Substrate (Fisher Scientific, Illkirch, France) with a ChemiCapt 3000 imaging system (Vilber Lourmat, Marne-la-Vallée France).

Séhédic D., Chourpa I., Tétaud C., Griveau A., Loussouarn C., Avril S., Legendre C., Lepareur N., Wion D., Hindré F., Davodeau F, & Garcion E. (2017). Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma. Theranostics, 7(18), 4517-4536.

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Quantifying Cas9 Protein Expression

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Total plant tissue originating from 10 IEs at different timepoints were frozen in LN2, ground to a fine powder by mortar and pestle, and resuspended in 200 μl 2x Laemmli Sample Buffer (Bio-Rad, Hercules, CA) with 2-mercaptoethanol. Samples were boiled for 5 min, and the total soluble protein extracts (25 μl or 40 μl per well) were separated on 4-20% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad, Hercules, CA) and subsequently transferred to a 0.45 μm nitrocellulose membrane (GVS, Sanford, ME). For detection of Cas9 protein, anti-CRISPR/Cas9 C-terminal mouse monoclonal antibody (SAB4200751; Sigma-Aldrich, St. Louis, MO) and ProSignal Dura ECL Reagent (Genesee Scientific, San Diego, CA) were used. PageRuler Plus Prestained Protein Ladder (10–250 kDa, Thermo Fisher, Waltham, MA) was used as a molecular weight marker, and Cas9 protein with a C-terminal double nuclear-localization tag (QB3 Macrolab, University of California, Berkeley) was used as a positive control.

Poddar S., Tanaka J., Running K.L., Kariyawasam G.K., Faris J.D., Friesen T.L., Cho M.J., Cate J.H, & Staskawicz B. (2023). Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.). Frontiers in Plant Science, 13, 1084700.

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Protein Interaction Analysis by Native PAGE

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Recombinant rEGFP-GBPL3 and rRFP-GBPL1 proteins were purified as described above. Reactions (20 μl) were incubated on ice for 30 min containing rEGFP-GBPL3 at a concentration of 37.5 ng/μl and increasing amount of rRFP-GBPL1 (0, 12.5, 37.5, 62.5 ng/μl) inassembly buffer (20mM HEPES-KOH [pH 7.9], 2.5 mM MgCl₂, 150 mM NaCl, 20% glycerol, 0.2% Triton X-100, 0.2 mM EDTA, 1 mM GTP). Reactions were loaded onto 4–20% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad, 4561096) and run at 100V constant voltage for 3 h at 4 °C. Native protein size marker (ThermoFisher, LC0725) was run alongside. Following gel electrophoresis, proteins were blotted to PVDF-membranes (Bio-Rad, 162–0177). After transfer, the membranes were blocked with 5% skim milk in TBST. The membranes were incubated overnight with primary antibodies in 5% BSA in TBST. After washing the membranes four times with TBST, HRP-conjugated secondary antibodies were applied for 1 h at room temperature. Detection was performed using Clarity Max Western ECL Substrate (Bio-Rad, 1705062). Reactions were also loaded onto SDS–PAGE gels in parallel as loading controls.

Huang S., Zhu S., Kumar P, & MacMicking J.D. (2021). A phase-separated nuclear GBPL circuit controls immunity in plants. Nature, 594(7863), 424-429.

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Western Blot Analysis of Exosomal Proteins

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Immunoblotting of Cellular Lysates

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Cells were lysed in TBS supplemented with 1% Triton X-100 and protease/phosphatase inhibitor cocktails (Roche Diagnostics), the insoluble material was cleared by centrifugation for 10 min at 20,000g. Immunoblotting was performed by standard methods using 4–15% Mini-PROTEAN TGX precast polyacrylamide gels and nitrocellulose membranes (Bio-Rad). Ponceau S staining of membranes was routinely used to assess equal sample loading and transfer efficiency. Blocking was performed in 5% milk in TBS with 0.1% Tween 20. Primary antibody incubations were performed in 5% BSA in TBS with 0.1% Tween 20. Signals were detected with HRP-conjugated secondary antibodies (Cell Signaling Technology) and either Super Signal West Pico or Femto chemiluminescent detection reagents (Thermo Fisher Scientific) on a VersaDoc imaging system (Bio-Rad). ImageJ was used to measure band intensities. Antibodies used in this study are listed in Table S4.

Table S4 Summary of antibodies used in this study.

Roczniak-Ferguson A, & Ferguson S.M. (2019). Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis. Life Science Alliance, 2(5), e201900358.

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