Western blots were used to examine shRNA knockdown efficiency. N2A cells were co-transfected with the constructs that express Flag-tagged FlRT3 or Flag-tagged CDKN1C and the corresponding shRNAs. After 24 h incubation, untransfected and transfected cells were harvested and lysed for protein extraction. Then the protein extract was run on 10% SDS PAGE gels. Proteins were then transferred to a PVDF membrane. The membranes were treated with blocking reagent (3% bovine serum albumin) for 1 h before incubated with the rabbit anti-RFP antibody (Abcam) overnight in a cold room. After several washes, the membranes were incubated with the secondary antibody (goat anti-rabbit immunoglobulins/HRP), washed again, and developed with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096). Images were collected using a Gel Doc XR+ Gel Documentation System (Bio-Rad) machine. The same membranes were then washed and incubated with the mouse Flag-m2 antibody (Sigma-Aldrich) overnight. On the second day, the membranes were washed, incubated with the secondary antibody (rabbit anti-mouse immunoglobulins/HRP), washed again, developed and exposed in the Gel Doc XR+ Gel Documentation System (Bio-Rad) machine. Details of the antibodies used are provided in Table S4 .
Gel doc xr gel documentation system
Manufactured by Bio-Rad
Sourced in United States, Germany
The Gel Doc XR+ Gel Documentation System is a compact, automated imaging system designed for the analysis of DNA, RNA, and protein samples separated by gel electrophoresis. It captures high-quality images of gels stained with various fluorescent dyes and provides advanced image analysis capabilities.
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Lab products found in correlation
Image lab software, by Bio-Rad (10 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Beyotime (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
T100 thermal cycler, by Bio-Rad (3 mentions)
Taq master mix, by Vazyme (3 mentions)
Sds page sample loading buffer, by Beyotime (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Sds page, by Beyotime (3 mentions)
Gelred nucleic acid gel stain, by Biotium (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Gelred 3 staining solution, by Biotium (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Mini trans blot cell system, by Bio-Rad (2 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Quantity one, by Bio-Rad (2 mentions)
Mini protean tetra cell, by Bio-Rad (2 mentions)
Clarity max ecl, by Bio-Rad (2 mentions)
125 protocols using gel doc xr gel documentation system
1
Evaluating shRNA Knockdown Efficiency
2
Immunoblotting Protocol for β-catenin Analysis
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Cell lysates used for immunoblotting were prepared in RIPA buffer (Abcam). Protein concentrations were analysed using the BCA Protein Assay Kit (Thermos Fisher Scientific). 20 ug of proteins were loaded and separated by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). After blocking with StartingBlock TBS (Thermo Fisher Scientific), membranes were incubated with primary monoclonal antibody against β-catenin (1:250, Thermo Fisher Scientific) and GAPDH (1:700, Thermo Fisher Scientific) at 4 °C overnight, washed and incubated with HRP-conjugated secondary antibody (1:1000, Thermo Fisher) at RT for 1 h. The membranes were then washed, stained using DAB Substrate Kit (Thermo Fisher Scientific) and visualised (Gel Doc XR + Gel Documentation System, Bio-Rad). The signal from each band was quantified by dedicated software for densitometric evaluation (Image Lab Software, Bio-Rad).
3
Western Blot for Protein Analysis
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Total proteins were extracted using SDS lysis buffer (4 %w/v SDS; 20 %v/v glycerol; 0.004 %w/v bromophenol blue; 0.125 M Tris-Cl, pH 6.8; 10 %v/v 2-mercaptoethanol) and sonication (50 kHz for 30 s; VibraCell X130PB, Sonics Materials) at 4 °C and subsequently denatured at 95 °C for 5 min. Proteins were separated on RunBlue 4-12 %w/v bis-tris polyacrylamide gels (Expedeon) and then transferred onto iBlot PVDF membranes (ThermoFisher) using the Wet/Tank Blotting Systems (Bio-Rad). Membranes were probed overnight at 4 °C in blocking solution containing the primary antibody. Primary antibodies used in this work were PARP14 (C-1) (Santa Cruz Biotechnology, sc-377150), Phospho-STAT1 (pSTAT1; Tyr701; 58D6) (Cell Signalling Technology, 9167 L), STAT1 (Cell Signalling Technology, 9172), MHC Class I H2 Kb (Abcam, ab93364), GAPDH (Proteintech, 60004-1-Ig), TAP1 (Cell Signalling Technology, 12341), TAP2 (Cell Signalling Technology, 12259; Santa Cruz Biotechnology, sc-515576), and PD-L1/B7-H1 (R&D Systems, AF1019-SP; Cell Signalling Technology, 13684). This was followed by incubation with the appropriate secondary antibody for 1.5 h at room temperature. Signals were developed using the Clarity Max Western ECL blotting substrate (Bio-Rad) and acquired on a Gel Doc XR+ Gel Documentation System (Bio-Rad). Images were analysed using Image Lab™ Software (version 3.0.1.).
4
Barnacle Settlement Protein Analysis
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Example 7
Adult barnacles were placed onto a nitrocellulose membrane (0.45 μm), fed and housed in ASW at room temperature for 72 hour settlement to allow for resettlement via cement deposition (
5
Quantifying Exosomal Protein Expression
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EV protein extracts (300 μg per medium type) were separated by Mini-PROTEAN TGXPrecast Gels (BioRad) and transferred to PVDF membranes by using Trans-Blot Turbo RTA Mini PVDF Transfer Kit (BioRad). The expression level of Syntenin and CD63 was evaluated by using goat polyclonal IgG Syntenin/SDCBP antibody (PA5-18595, Invitrogen/Thermo Fisher Scientific) and mouse monoclonal IgG exosome—anti-CD63 antibody (10628D, Invitrogen), respectively. An equal loading in the lanes was evaluated by mouse monoclonal IgG β-actin antibody (sc-81178, Santa Cruz Biotechnology). The level of analyzed proteins was subsequently detected with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (H+L) secondary antibody (R21459, Invitrogen) or goat anti-mouse IgG, IgM (H+L) secondary antibody (31,444, Invitrogen). All antibodies were used according to manufacturer’s protocols. The membranes were developed with Luminata Crescendo Western HRP Substrate (Merck/Millipore, Darmstadt, Germany) and imaged by Gel Doc XR+ Gel Documentation System (Bio-Rad).
6
Genomic DNA Isolation and PCR Amplification
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Total genomic DNA was isolated from leaf tissue of test lines using Cetyl Trimethyl Ammonium Bromide (CTAB) method25 . Polymerase chain reaction (PCR) was performed in a thermal cycler (Applied Biosystem Veriti, California, USA) using a total reaction mix of 10 μl by adding 25–30 ng of genomic DNA. 5 pmol of each of the two primers (synthesized from Sigma-Aldrich Inc., St. Louis, MO, USA), 0.2 mM dNTPs, 1.5 mM MgCl2, (MBI, Fermentas, Vilnius, Lithuania) and 0.5 U of Taq polymerase (Bangalore Genei, Bangalore, India). PCR amplification was performed by one cycle of denaturation at 95 °C for 4 min, followed by 35 cycles at 95 °C for 40 s, 55 °C for 40 s and 72 °C for 1 min, with a final extension of 72 °C for 10 min. The PCR amplified products were resolved on 3.5% Metaphor Agarose gel containing 0.1 mg/mL of ethidium bromide (Amresco, Solon, OH, USA) along with a DNA size standard 50 bp ladder (MBI, Fermentas, Vilnius, Lithuania) and visualized on ultraviolet trans-illuminator (Gel Doc XR + Gel Documentation system, Bio-Rad Laboratories Inc.,U.S.A).
7
Western Blot Protein Detection
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Proteins were separated by 8, 12, or 16% SDS–PAGE and transferred onto Nitrocellulose membranes. After incubation with primary antibody overnight and secondary antibody (goat-anti-rabbit immunoglobulin G [IgG] horseradish peroxidase [HRP] [1:10,000] or goat anti-mouse IgG HRP [1:5000; Bio-Rad]) for 1 h at room temperature, signals were visualized in a Bio-Rad Gel Doc XR+Gel Documentation System.
8
EMSA Analysis of 5'-UTR and Nsp1 Binding
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Samples for EMSA were prepared by diluting the appropriate volumes of concentrated stock solutions (Table S3 ) of 5′-UTR (7.7 μM) and Nsp1 (38 and 152 μM) in EMSA buffer (50 mM sodium phosphate (pH 6.5), 50 mM NaCl, 2 mM EDTA, 2 mM DTT; prepared with nuclease-free water) to a total volume of 10 μL, followed by incubation for 30 mins at room-temperature. Four different concentrations of 5′-UTR were used (0.3, 0.6, 1.25 & 2.5 μM), with five samples prepared at each 5′-UTR concentration (corresponding to 0-, 5-, 10-, 20- and 40-fold excess Nsp1), giving 20 samples in total. 2.5 μL 5x loading-dye (50 mM Tris–HCl (pH 7.5), 0.25% xylene cyanol, 0.25% bromophenol blue, 30% glycerol) was added to each sample just prior to loading onto a 5% polyacrylamide gel, which had been pre-run for 1 h with TBE running-buffer (100 mM Tris-HCl (pH 8.3), 100 mM boric acid, 2 mM EDTA). After sample-loading, the gel was run overnight (with the same running-buffer) at 4 °C and a current of 2 mA. The gel was stained in a solution of ethidium bromide (0.25 μg/mL, 2 mins incubation), washed three times in water and then visualized using a Gel Doc XR+ gel documentation system (Bio-Rad).
9
Protein Breakdown Analysis via SDS-PAGE
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The homogenized digests were examined for the breakdown of specific proteins using reduced-Tricine-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Chian et al. [23 (link)]. The digests were mixed with tricine sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), then 25 µL of each sample was loaded into individual wells (16.5% gradient Tricine gels, CriterionTM Gel, Bio-Rad Laboratories, Hercules, CA, USA) at a protein concentration of 1 mg/mL. Gels were run using a CriterionTM cell (Bio-Rad Laboratories, Hercules, CA, USA) at 125 V and then stained with Bio-safe Coomassie blue stain (Bio-Rad Laboratories, Hercules, CA, USA). The gel was scanned with a Gel Doc XR + Gel Documentation System (Bio-Rad Laboratories, Hercules, CA, USA), followed by analysis using Image LabTM software (version 6.1.0, Bio-Rad Laboratories, Hercules, CA, USA).
10
Topoisomerase II Inhibition Assay
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The inhibition of yeast topoisomerase II was analysed according to the relaxation assay kit from Inspiralis (Inspiralis Limited, Norwich, UK). Briefly, 250 ng of supercoiled pBR322 DNA (Thermo Fisher Scientific, Waltham, MA, USA), 1 mM ATP (Inspiralis Limited, UK), and 1–200 μM of the analysed compound were mixed with reaction buffer (1 mM Tris-HCl (pH 7.9), 10 mM KCl, 0.5 mM MgCl2, 0.2 % (by volume) glycerol). The reaction was initiated by the addition of an enzyme, allowed to proceed at 30 °C for 30 min and terminated by addition of 40% (m/V) sucrose, 100 mM Tris-HCl pH 8, 10 mM EDTA, and 0.5 mg mL−1 Bromophenol Blue. Two-step extraction with chloroform:isoamyl alcohol (24:1, by volume) and butanol water was performed and mixtures were analysed on 1% agarose gel in 1 × TAE buffer, 3h, 4.5 V cm−1. Gel was stained in GelRed 3 × staining solution (Biotium, Fremont, CA, USA) for 30 min and photographed with Gel Doc XR+Gel Documentation System (Bio-Rad: Hercules, CA, USA).
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