Polyacrylamide gel

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4–12% polyacrylamide gel is a laboratory equipment used for electrophoresis. It is a gel matrix composed of polyacrylamide with a gradient concentration ranging from 4% to 12%. This gel is designed to separate and analyze biomolecules, such as proteins, based on their size and charge.

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4–20% Tris–Glycine polyacrylamide precast gels Gradient polyacrylamide Bis-Tris gels 6% non-denaturing polyacrylamide gels Bolt Bis‐Tris Plus 4–12% precast polyacrylamide gels Novex precast 4–20% tris-glycine polyacrylamide gels Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels Nu-PAGE polyacrylamide gels 4–12% SDS-polyacrylamide pre-cast gels NuPAGE precast polyacrylamide gels Tris-HEPES-SDS polyacrylamide gels

169 protocols using polyacrylamide gel

Western Blot Analysis of Protein Expression

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Protein was prepared in NuPAGE Sample Buffer and Reducing Agent (Life Technologies, Carlsbad, CA, USA) using 10 μg (estrogen-related blots), 65 μg (V5 blot, T47D-ELF5-isoform 2-V5) or 25 μg (V5 blots, all other lines) per lane. Samples were separated on precast 15-well 4–12 % Bis-Tris (estrogen-related blots) or 10-well 10 % Bis-Tris (V5 blots) polyacrylamide gels (Life Technologies), transferred to polyvinylidene fluoride membrane, blocked in 5 % skim milk, and incubated overnight at 4 °C in primary antibody. Secondary horseradish peroxidase–conjugated antibody was added 1:2000 in 5 % skim milk (anti-mouse, NA931V, anti-rabbit, NA934V; GE Healthcare Life Sciences, Little Chalfont, UK). Proteins were detected using enhanced chemiluminescence solution (Western Lightning Plus; PerkinElmer, Waltham, MA, USA) and x-ray film (Fujifilm, Tokyo, Japan). Primary antibodies used were anti-V5 (sc-58052, 1:500–1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-transducin-like enhancer of split 1 (anti-TLE1) (ab183742, 1:1000; Abcam, Cambridge, UK), anti-ERα (sc-8005, 1:1000; Santa Cruz Biotechnology), anti-Forkhead box A1 (anti-FOXA1) (sc-101058, 1:1000, Santa Cruz Biotechnology), and anti-β-actin (AC-15, 1:20,000; Sigma-Aldrich, St. Louis, MO, USA).

Piggin C.L., Roden D.L., Gallego-Ortega D., Lee H.J., Oakes S.R, & Ormandy C.J. (2016). ELF5 isoform expression is tissue-specific and significantly altered in cancer. Breast Cancer Research : BCR, 18, 4.

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Etioplast Protein Extraction and Analysis

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A defined amount of etioplasts (10⁸) were lysed and washed two times in TMK and solubilized in a detergent mix composed of 0.38% (w/v) n-dodecyl-β-d-maltoside, 0.64% (w/v) digitonin and 0.006% (w/v) lithium dodecyl sulfate (LDS), as described [17 (link)]. All steps were performed on ice and non-soluble material was separated by centrifugation (4°C, 16100 rcf, 10 min). Soluble protein extracts were separated by LDS-Native (LN) PAGE on 7.5% (w/w) [17 (link)] or 3–12% polyacrylamide gels (Novex, Life technologies, California USA). The cathode buffer was supplemented with 74 μM LDS and pigment binding proteins were detected by fluorescence scanning (filter 670 BP30) using a HeNe laser excitation at 633 nm in a Typhoon scanner (GE Healthcare, Buckingham, GB).

Mork-Jansson A., Bue A.K., Gargano D., Furnes C., Reisinger V., Arnold J., Kmiec K, & Eichacker L.A. (2015). Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley. PLoS ONE, 10(7), e0133145.

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Traction Force Mapping of Stellate Cells

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Poly acrylamide gels containing 0.5 μm diameter fluorescent beads (Life Technologies, Carlsbad, CA, USA) were prepared with a Young’s moduli of 5 kPa using a ratio 7.5:0.175 of acrylamide (40% w/v; Bio-Rad) to bis-acrylamide (2% w/v; Bio-Rad) and then coated with 0.1 mg/mL of rat tail type I collagen (Corning) [55 (link),56 (link)]. HSteCs (20,000 cells) were grown in 35-mm dishes for 24 h, and then large EVs from TJU-UM001 cells (100 µg) were deposited onto adherent stellate cells. After 24 h, phase contrast images of single HSteCs and fluorescent images of the bead field at the surface of the polyacrylamide gel were acquired (Nikon Eclipse Ti microscope, NIS-Elements software) [56 (link),57 (link)]. Cells were removed from the polyacrylamide gel using 0.25% trypsin/EDTA, and a fluorescent image of the bead field was acquired after cell removal [56 (link),57 (link)]. Bead displacements between stressed (+EVs) and null (w/o EVs) states were analyzed for 25 cells per condition using the LIBTRC library to obtain traction stress maps (Pa) and traction force values (nN) [56 (link),58 (link)].

Piquet L., Coutant K., Mitchell A., Ben Anes A., Bollmann E., Schoonjans N., Bérubé J., Bordeleau F., Brisson A, & Landreville S. (2022). Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment. Cells, 11(23), 3828.

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Calcitriol and EB1089 Assay Protocol

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Calcitriol (1,25-(OH)₂ vitamin D₃) was purchased from Cayman Chemical (Cat #71820). EB1089 was purchased from R&D Systems (Cat #3993). RIPA lysis buffer (Cat #R0278) and protease and phosphatase cocktails (Cat #P8340, Cat #P5726), were all purchased from Sigma Aldrich. All antibodies were purchased from Cell Signaling Technology. FBS was purchased from Seradigm (Cat #97068–085). IL-2 was purchased from Miltenyi Biotec (Cat #130-097-743). Clarity enhanced chemiluminescence (ECL) reagent (Cat #170–5061) and PVDF membrane and paper stacks (Cat #170–4274), were purchased from BioRad. RPMI 1640 (Cat #10–040-CV), Pierce bicinchoninic acid (BCA) protein assay kit (Cat #PI23225), and SuperSignal West Femto Maximum Sensitivity Substrate (Cat #34096) were purchased from ThermoFisher Scientific. ReBlot Plus Strong Antibody Stripping Solution 10X was purchased from Millipore Sigma (Cat #2504). Polyacrylamide gels (4–12%) were purchased from Life Technologies (Cat #NW04125BOX). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Cell Proliferation Colorimetric Assay Kit was purchased from BioVision (Cat #K300–2500). Molecular weight markers used in western blotting were SeeBlue Plus2 Pre-Stained Standard (ThermoFisher Scientific) and Flash Protein Ladder #51121659 and #51121660 (WorldWide Life Sciences Division).

Olson K.C., Kulling P.M., Signorelli R., Hamele C.E., Olson T.L., Conaway M.R., Feith D.J, & Loughran TP J.r. (2018). Vitamin D pathway activation selectively deactivates signal transducer and activator of transcription (STAT) proteins and inflammatory cytokine production in natural killer leukemic large granular lymphocytes. Cytokine, 111, 551-562.

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Cell Lysate Fractionation and Immunoblotting

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Total protein, chromatin, cytosolic and nuclear soluble cell lysates were purified as described [45 (link),46 (link)], and separated using polyacrylamide gels (Invitrogen) prior to transfer to PVDF membranes. Primary antibodies were Cdt1 (F-6), cyclin E1 (HE12), Cdc6 (180.2), CDK2 (M2), cyclin A2 (BF683) and GAPDH (6C5) from Santa Cruz Biotechnology, cyclin E2 (EP454Y) from AbCam and MCM2 (#3619), MCM7 (#3735), ORC6 (#4737) and Ddb1 (#5428) from Cell Signaling. Additional antibodies, chemiluminescence and densitometry are described in [18 (link)]. Immunoprecipitation antibodies were cyclin E1(C-19) from Santa Cruz Biotechnology and cyclin E2 (AbCam, [EP454Y]).
Densitometry of western blots was performed using ImageJ [47 ].

Lee C., Fernandez K.J., Alexandrou S., Sergio C.M., Deng N., Rogers S., Burgess A, & Caldon C.E. (2020). Cyclin E2 Promotes Whole Genome Doubling in Breast Cancer. Cancers, 12(8), 2268.

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Nuclear Protein Extraction and ISGF3/NF-κB Gel Shift Assay

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Nuclear protein extracts were prepared from cells after treatment with LPS or IFN-β, according to a modification [19 (link)] of the original method described by Dignam et al. [20 (link)]. A double-stranded oligonucleotide, based on a DNA sequence in the promoter of the human ISG15 gene, was used as a probe for ISGF3 in the gel shift assays [21 (link)]. This oligonucleotide contains an ISRE that has a high affinity for ISGF3 complexes. The NF-κB probe, obtained from Promega Corp. (Madison, WI, USA; catalog no. E3292), was a consensus oligonucleotide based on an NF-κB binding site in the Igκ gene [22 (link)]. These probes were end-labeled with [γ-³²P]-ATP using T4 polynucleotide kinase, and binding reactions were performed [23 (link)]. A portion of each binding-reaction mixture (8 μL per sample) was electrophoresed on nondenaturing, 6% polyacrylamide gels (Invitrogen, Carlsbad, CA, USA), using 0.25× Tris-borate-EDTA buffer (22 mM Tris-HCl [pH 8.0], 22 mM borate, and 0.5 mM EDTA). The gels were subsequently dried and visualized by autoradiography.

Sheikh F., Dickensheets H., Gamero A.M., Vogel S.N, & Donnelly R.P. (2014). An essential role for IFN-β in the induction of IFN-stimulated gene expression by LPS in macrophages. Journal of Leukocyte Biology, 96(4), 591-600.

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STAT6 and STAT1 Binding Assay Protocol

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Nuclear protein extracts were prepared from cells after treatment with IL-4 or
IL-13 as previously described [25 (link)]. A
double-stranded oligonucleotide, based on a DNA sequence in the promoter of the human IL-1
receptor antagonist gene, IL1RN, was used as a probe for STAT6 in the gel
shift assays [26 (link)]. This oligonucleotide contains a
STAT-binding element (SBE1) that has a high affinity for STAT6 complexes. The GAS probe
used to measure STAT1 activity was derived from a sequence in the promoter of the human
IgG FcγR1 gene, FCGR1a [27 (link)]. These probes were end-labeled with [γ-³²P]-ATP using T4
polynucleotide kinase, and binding reactions were performed as described previously [25 (link)]. A portion of each binding reaction mixture (8
µL per sample) was electrophoresed on non-denaturing, 6% polyacrylamide
gels (Invitrogen) using 0.25×
Tris-borate-ethylenediamine-N,N,N’,N’-tetraacetic acid buffer (22 mM
Tris-HCl, pH 8.0, 22 mM borate, and 0.5 mM
ethylenediamine-N,N,N’,N’-tetraacetic acid). The gels were subsequently
dried and visualized by autoradiography.

Sheikh F., Dickensheets H., Pedras-Vasconcelos J., Ramalingam T., Helming L., Gordon S, & Donnelly R.P. (2015). The Interleukin-13 Receptor-α1 Chain Is Essential for Induction of the Alternative Macrophage Activation Pathway by IL-13 but Not IL-4. Journal of Innate Immunity, 7(5), 494-505.

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SDS-PAGE Analysis of GMMA Proteins

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GMMA were denatured for 10 min at 95 °C in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 2 % (wt/vol) SDS and subsequently loaded onto 12 % (wt/vol) polyacrylamide gels (Invitrogen). Gels were run in 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer (BioRad) and were stained with brilliant Blue G-colloidal Coomassie (Sigma-Aldrich B2025) according to the manufacturer’s instructions with slight modifications. Briefly, after 30 min fixation with 40 % (vol/vol) methanol and 7 % (vol/vol) acetic acid in water, gels were stained overnight using 40 mL of reconstituted brilliant Blue G-colloidal Concentrate (0.1 % (wt/vol) Brilliant Blue G, 0.29 M phosphoric acid, 16 % saturated ammonium sulfate) and 15 mL of methanol. Gels were destained using 30 % methanol in water (vol/vol) for 2 h.

Rossi O., Maggiore L., Necchi F., Koeberling O., MacLennan C.A., Saul A, & Gerke C. (2014). Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA). Molecular Biotechnology, 57(1), 84-93.

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Protein Extraction and Western Blot

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Proteins were extracted in RIPA buffer in presence of phosphatase and protease inhibitors. Proteins were then seperated on polyacrylamide gels (Invitrogen). Phospho Akt (Ser473) and Akt antibodies were from Cell Signaling 5St Quentin Yvelines, France). α-Tubulin antibody was from Santa Cruz (Heidelberg, Germany) [1] (link).

Patsouris D., Cao J.J., Vial G., Bravard A., Lefai E., Durand A., Durand C., Chauvin M.A., Laugerette F., Debard C., Michalski M.C., Laville M., Vidal H, & Rieusset J. (2014). Insulin Resistance is Associated with MCP1-Mediated Macrophage Accumulation in Skeletal Muscle in Mice and Humans. PLoS ONE, 9(10), e110653.

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Quantitative Western Blot Analysis

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After washing with ice-cold PBS, cells were lysed on ice in 100μL RIPA buffer containing 100mM PMSF. Cell lysates were collected and centrifuged at 14,000 rpm for 10 mins at 4°C. The protein lysates were then mixed with 5×loading buffer and denatured by boiling for 5 mins at 100°C, separated on 10% polyacrylamide gels (Invitrogen), and transferred to PVDF (polyvinylidene fluoride) membranes. Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBS-T) for 2.5 hrs. Thereafter, membranes were incubated overnight in antibodies targeting RIPK1 (Abcam, Cambridge, UK, 1:1000), AP-1 (Abcam, Cambridge, UK, 1:1000), P-AP-1 (Abcam, Cambridge, UK, 1:1000), JNK (Abcam, Cambridge, UK, 1:1000), P38MAPK (Abcam, Cambridge, UK, 1:1000), ERK1/2 (Abcam, Cambridge, UK, 1:1000), E-cadherin (Abcam, Cambridge, UK, 1:500) N-Cadherin (Abcam, Cambridge, UK, 1:1000) and VEGF-C (Santa Cruz, New York, USA, 1:1000) at 4°C. After conjugating with respective HRP-coupled secondary antibodies, the protein–antibody complexes were detected using chemiluminescence (Life Technologies, USA) and recorded on Hyperfine-ECI detection film. Target protein expression was semi-quantified relative to GAPDH (loading control) expression.

Li C.Z., Lin Y.X., Huang T.C., Pan J.Y, & Wang G.X. (2019). Receptor-Interacting Protein Kinase 1 Promotes Cholangiocarcinoma Proliferation And Lymphangiogenesis Through The Activation Protein 1 Pathway. OncoTargets and therapy, 12, 9029-9040.

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