Tris glycine gel

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Tris-Glycine gels are a type of polyacrylamide gel used for electrophoresis, a technique for separating and analyzing proteins. These gels consist of a Tris-Glycine buffer system and are commonly used in SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) applications.

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461 protocols using tris glycine gel

Quantifying Yeast Catalase Expression and Activity

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The expression and activity of yeast catalase were measured following
the methods described previously^{48 (link),49 (link)}. In brief,
yeast cells were cultured in 10 mL SCE media for 14–16 h to
mid-exponential phase. Cells were lysed using zirconium oxide beads in a Bullet
Blender (Next Advance)^{19 (link)}.
Protein concentrations were determined by the Bradford method (Bio-Rad) and 100
μg protein were subjected to SDS–PAGE on 10% Tris-glycine gels
(Invitrogen). Following incubation with anti-Ctt1p^{50 (link)} or anti-GAPDH (Genetex) primary
antibodies and a goat anti-rabbit secondary antibody conjugated to a 680 nm
emitting fluorophore (Biotium), the blots were imaged on a LI-COR Odyssey
Infrared imager^{19 (link)}.
To detect the catalase activity, 15 μg protein lysates were
analysed by PAGE on a 10% Tris-glycine gel (Invitrogen). After electrophoresis,
an in-gel activity stain was utilized to measure catalase activity^{48 (link),51 (link)}. In brief, the catalase staining solution was prepared
by mixing 1 part dopamine (20 mg ml⁻¹) and 1 part
para-phenylenediamine (3.5 mg ml⁻¹) in 0.2 M potassium
phosphate buffer, pH 8, 1 part 15% H₂O₂, and 2 parts DMSO
in the order listed. The staining solution was added directly to the gel and
allowed to stain for 2 min, followed by imaging.

Sun F., Zhao Z., Willoughby M.M., Shen S., Zhou Y., Shao Y., Kang J., Chen Y., Chen M., Yuan X., Hamza I., Reddi A.R, & Chen C. (2022). HRG-9 homologues regulate haem trafficking from haem-enriched compartments. Nature, 610(7933), 768-774.

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SDS-PAGE Analysis of SOSIP Trimers

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Two micrograms of SOSIP trimer or 3.2 µg of SOSIP-I53-50NP (which is equivalent to 2 µg of trimer) were loaded on a 4–12% Tris-Glycine gel or 6–18% Tris-Glycine gel, respectively (both from Invitrogen). SDS-PAGE (polyacrylamide gel electrophoresis) was performed as described previously^{7 (link)}.

Brouwer P.J., Antanasijevic A., Berndsen Z., Yasmeen A., Fiala B., Bijl T.P., Bontjer I., Bale J.B., Sheffler W., Allen J.D., Schorcht A., Burger J.A., Camacho M., Ellis D., Cottrell C.A., Behrens A.J., Catalano M., del Moral-Sánchez I., Ketas T.J., LaBranche C., van Gils M.J., Sliepen K., Stewart L.J., Crispin M., Montefiori D.C., Baker D., Moore J.P., Klasse P.J., Ward A.B., King N.P, & Sanders R.W. (2019). Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle. Nature Communications, 10, 4272.

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Optimized Western Blot Analysis Protocol

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Western blot analysis was performed as described previously [19 (link)]. Briefly, total cell lysates as well as mitochondrial and cytosolic fractions were purified and separated by 4-20% Tris-Glycine Gel (Invitrogen, Carlsbad, CA) electrophoresis. For active caspase-3, PARP, and VentX analysis, total cells were extracted and separated by 4-20% Tris-Glycine Gel (Invitrogen) electrophoresis. Antibodies used included GFP (Santa Cruz Biotech, Santa Cruz, CA), Histone-1 (Ab-1, NeoMarkers, Fremont, CA), β-actin (Sigma), active caspase-3 (BD Biosciences), and PARP (Cell Signaling Technology, Boston, MA). Appropriate horseradish peroxidase-conjugated secondary antibodies were used to detect bound primary antibody antigen complex and developed with Western Lightning® Western Blot Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA).

Gao H., Wu B., Le Y, & Zhu Z. (2016). Homeobox protein VentX induces p53-independent apoptosis in cancer cells. Oncotarget, 7(26), 39719-39729.

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Western Blot Analysis of STAT1 and Mx1

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Cell lysates were prepared using mammalian protein extraction reagent M-PER (Thermo Fisher Scientific, Waltham, MA) with Protease and Halt™ phosphatase inhibitor cocktails (Thermo Fisher Scientific) using an equal number of cells per sample. Samples were analyzed by SDS-PAGE using 10–20% Tris-Glycine gels (Thermo Fisher Scientific) under reducing conditions. As a molecular weight marker, protein ladder (cat# 7727S) from Cell Signaling Technology (Danvers, MA) was used. Nitrocellulose membranes and iBlot™ transfer system (Thermo Fisher Scientific) were used for Western Blot analysis. All other reagents for Western Blot analyses were purchased from Thermo Fisher Scientific. Membranes were blocked with nonfat dry milk (BIO-RAD, Hercules, CA) for 1 h followed by incubation with primary antibodies against STAT1, pSTAT1 (pY701, BD Transduction Lab, San Jose, CA), or Mx1 (gift from O. Haller, University of Freiburg, Freiburg, Germany) O/N at 4 °C. Secondary goat anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. SuperSignal West Femto Maximum Sensitivity Kit (Thermo Fisher Scientific) was used to develop membranes, and images were taken using LAS-3000 Imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA).

Chiang A.W., Li S., Kellman B.P., Chattopadhyay G., Zhang Y., Kuo C.C., Gutierrez J.M., Ghazi F., Schmeisser H., Ménard P., Bjørn S.P., Voldborg B.G., Rosenberg A.S., Puig M, & Lewis N.E. (2019). Combating viral contaminants in CHO cells by engineering innate immunity. Scientific Reports, 9, 8827.

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Western Blot Analysis of RpoA-NTD and ProQ

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Cell lysates from β-gal assays were normalized based on pre-lysis OD₆₀₀. Lysates were mixed with 6× Laemmli loading dye with PopCulture Reagent (EMD Millipore Corp), boiled for 10 min at 95°C and electrophoresed on 10–20% Tris–glycine gels (Thermo Fisher) in 1× NuPAGE MES Running Buffer (Thermo Fisher). Proteins were transferred to PVDF membranes (BioRad) using a semi-dry transfer system (BioRad Trans-blot Semi-Dry and Turbo Transfer System) according to manufacturer's instructions, and probed with 1:10 000 primary antibody (anti-RpoA-NTD; Neoclone or anti-ProQ; kindly provided by G. Storz) overnight at 4°C and then a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse IgG or anti-rabbit IgG; Cell Signaling, 1:10 000). Note that, throughout the paper, ‘anti-ProQ’ is written out rather than using the standard abbreviation of ‘α-ProQ.’ This is to avoid confusion with the fusion protein we call ‘α-ProQ’ consisting of the NTD of RpoA (α) fused in frame to ProQ. Chemiluminescent signal from bound peroxidase complexes was detected using ECL Plus western blot detection reagents (BioRad) and a c600 imaging system (Azure) according to manufacturer's instructions.

Pandey S., Gravel C.M., Stockert O.M., Wang C.D., Hegner C.L., LeBlanc H, & Berry K.E. (2020). Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ. Nucleic Acids Research, 48(8), 4507-4520.

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Western Blot Analysis of β-Galactosidase

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Cell lysates from β-gal assays were normalized based on OD600 with LB plus PopCulture Reagent. Lysates were mixed with 6× Laemmli loading dye, boiled for 10 min at 95°C and electrophoresed on 10%–20% Tris–glycine gels (Thermo Fisher) in 1× NuPAGE MES Running Buffer (Thermo Fisher). Proteins were transferred to PVDF membranes (Bio-Rad) using a semidry transfer system (Bio-Rad Trans-blot Semidry and Turbo Transfer System) according to the manufacturer’s instructions. Membranes were probed with 1:10,000 primary antibody anti-ProQ overnight at 4°C and then a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG; 1:10,000). Chemiluminescent signal was detected using ECL Plus Western blot detection reagents (Bio-Rad) and a c600 imaging system (Azure) according to the manufacturer’s instructions.

Stein E.M., Wang S., Dailey K.G., Gravel C.M., Wang S., Olejniczak M, & Berry K.E. (2023). Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ. RNA, 29(11), 1772-1791.

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Immunoblotting of Cell Signaling Proteins

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Cells were trypsinized and washed in cold PBS before being lysed in 2X Laemmli sample buffer containing 0.5% beta-mercaptoethanol, 1% each of Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 2, and Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, P8340, P5726, P0044, respectively). Samples were run on 4-20% Tris-Glycine gels (Life Technologies) and transferred to 0.45 μm PVDF-FL membrane (Millipore). Membranes were blocked in 2% milk solution and probed with the following antibodies: NMIIB (Biolegend, rabbit pAb; 1:1000 cat #909901), GAPDH (Santa Cruz Biotechnology rabbit pAb; 1:5000). EGFR (Cell Signaling Technology, rabbit pAb; 1:1000). Blots were analyzed with LiCor Odyssey CL imaging system.

Thomas D., Thiagarajan P.S., Rai V., Reizes O., Lathia J, & Egelhoff T. (2016). Increased cancer stem cell invasion is mediated by myosin IIB and nuclear translocation. Oncotarget, 7(30), 47586-47592.

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Optimized Western Blot Protocol for Protein Analysis

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For western blot analysis, we used a procedure allowing efficient recovery of proteins from all samples, i.e., whole cells or subcellular fractions. To this end, for all samples prepared by procedures 1–4, protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) were added and samples underwent 3 freeze/thaw cycles to disrupt membranes. Purified His-tagged Rep68 protein (500 ng)³⁰ (link) was then spiked in each sample, before the addition of 4 sample volumes of ice-cold acetone and overnight precipitation at −20°C. Proteins were then pelleted at 16,000 × g for 15 min at 4°C and resuspended in 150 μL of 1× Laemmli buffer. SDS-PAGE was conducted using 25 μL of each protein sample, which was denatured at 95°C for 5 min before loading on 10% Tris-glycine gels (Life Technologies). Gels were transferred on nitrocellulose membranes and probed with antibody against lamin B, α-tubulin, ATP-synthase, or Rep proteins. Membranes were then incubated with HRP-conjugated secondary antibodies followed by chemiluminescence detection with ECL substrate (Thermo Scientific).

François A., Bouzelha M., Lecomte E., Broucque F., Penaud-Budloo M., Adjali O., Moullier P., Blouin V, & Ayuso E. (2018). Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls. Molecular Therapy. Methods & Clinical Development, 10, 223-236.

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Extraction of Proteins from FFPE Tissue

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Western blot analyses were performed as previously described (Nandan et al., 2010 (link)). Proteins were extracted from 20 µm paraffin embedded tissue sections using a previously established protocol (Shi et al., 2006 (link)). Tissue sections were deparaffinized using xylene with the addition of 7.5% methanol and were then centrifuged and the pellet dried in a fume hood for 3 min. The pellets were then resuspended in 20 mM Tris-HCl (pH 7.5) containing 2% SDS and the suspension heated in a 100 °C heat block for 20 min. Subsequently, the samples were incubated in a 60 °C oven for 2 hr. Protein content was measured and equal amounts of samples were loaded onto Tris-Glycine gels (Life Technologies, CA, USA). Proteins were transferred to nitrocellulose membranes (BioRad, CA, USA) and probed with appropriate primary antibodies. Blots were then washed and secondary antibodies applied at appropriate concentrations. Protein bands were subsequently visualized on film upon chemiluminescent detection.

Nandan M.O., Ghaleb A.M., Liu Y., Bialkowska A.B., McConnell B.B., Shroyer K.R., Robine S, & Yang V.W. (2014). Inducible Intestine-specific Deletion Of Krüppel-Like Factor 5 Is Characterized By A Regenerative Response In Adult Mouse Colon. Developmental biology, 387(2), 191-202.

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Characterizing α-crystallin-histone interactions

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Supernatants and pellets were obtained by centrifuging (10,000 rpm for 30 min) the complexes of α-crystallin (50 μM) and histones (0.4–6.42 μM) after a 1-h incubation at 37 °C. Thirty microliters of electrophoresis sample buffer (Novex LC2676 Tris-glycine-SDS buffer; Life Technologies) was added to each pellet, and 20 μl was added to the lanes of 10–20% Tris-glycine gels (Life Technologies). Prestained molecular weight markers (Invitrogen) were used on all gels. After the electrophoresis, the gels were stained with Coomassie blue or transferred to polyvinylidene difluoride membranes and stained with Revert protein stain (LI-COR Biosciences, Lincoln, NE, USA) and visualized on an Odyssey analyzer (LI-COR).

Hamilton P.D, & Andley U.P. (2018). In vitro interactions of histones and α-crystallin. Biochemistry and Biophysics Reports, 15, 7-12.

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