Using a lysis buffer (0.1 % SDS, 1 %TritonX‑100, 1.5 M, 1 mM EDTA, 20 mM Tris‑HCl (pH 7.4), 1 % phenylmethanesulfonyl fluoride, and 1.5 M NaCl), the cells were lysed and total protein was purified through a 16,000 g centrifugation at 4 °C for 10 min. Using the DC protein test from Bio-Rad, the protein concentration was calculated. A constant voltage of 80 mV for 30 min, followed by 120 mV for 120 min, was used to separate 30 µg of protein from each group by electrophoresis, which was subsequently transferred to PVDF membranes using the 100 mV for 120 min Bio-Rad TransBlot system (Bio Rad Laboratories, Inc.). 5 % non-fat dried milk dissolved in 10 mM Tris HCl, 0.2 % Tween-20 (TBST), and 0.15 M NaCl were then used to block the membrane for 2 hours at room temperature. The membrane was incubated with the primary antibodies against Bax, Bcl-2, caspase-3, MMP-3, MMP-13, and β-Actin for a whole night at 4 °C. The membrane was incubated with m-IgGκBP-HRP and mouse anti-rabbit IgG-HRP secondary antibodies at room temperature for 2 hours after being rinsed with TBST three times for 5 minutes. The membrane was treated with an ECL chemiluminescence kit and tested with Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad) for analysis. ImageJ v1.8.0 (National Institutes of Health) software was used to densitometry quantification of the images.
Trans blot system
Manufactured by Bio-Rad
Sourced in United States, Canada, Germany
The Trans-Blot system is a laboratory equipment designed for protein transfer. It facilitates the transfer of proteins from a gel to a membrane, allowing for further analysis and detection. The core function of the Trans-Blot system is to enable the efficient transfer of proteins from one medium to another.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (6 mentions)
Chemidoc mp system, by Bio-Rad (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Fast cast tgx stain free, by Bio-Rad (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
Pi 1000, by Vector Laboratories (3 mentions)
M7522, by Merck Group (3 mentions)
Hrp conjugated goat antirabbit igg, by Vector Laboratories (3 mentions)
Clarity max ecl, by Bio-Rad (3 mentions)
Cfx software, by Bio-Rad (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
β actin, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Odyssey system, by LI COR (2 mentions)
Ecl gels, by GE Healthcare (2 mentions)
97 protocols using trans blot system
1
Western Blot for Apoptosis Markers
2
Western Blot Analysis of OXPHOS and AMPK
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HepG2 cells were collected in Laemmli buffer and protein samples were transferred to membranes by electrotransfer from sodium dodecyl sulfate polyacrylamide gel (10%) using a Bio-Rad Trans-Blot system (Bio-Rad Laboratories, Inc., Tokyo, Japan). Membranes were blocked for 1 h at room temperature in 5% nonfat milk diluted in Tris buffered saline solution containing 0.1% Tween 20 (TBS-T). Then the membranes were washed in TBS-T and incubated with primary antibodies for OXPHOS, AMPK, p-AMPK and beta-actin (Sigma-Aldrich) overnight, under stirring at 4 °C. After that, the membranes were incubated with secondary antibodies for 2 h at room temperature. Finally, the detection of the blot was realized with a chemiluminescence kit (substrate ECL Western ClarityTM, Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Drosophila and HeLa Cells
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Drosophila S2 cell samples were boiled for 10′ in loading buffer separated on 6% (Spt6) or 10% (Co-IPs) SDS-PAGE gels and processed for western blotting using mouse α GFP (1:2000, Clone 71; D. van Essen), monoclonal mouse α Spt6 (25C6, Helmholtz Zentrum München, IgG2b 1:500) was raised against Spt6 amino acid residues 215–230 DYDDFSKYEEDDYEDD, rabbit α CENP-A (1:5000, ab10887, Abcam), mAB α Flag (Clone M2; 1:10000, F1804, Sigma-Aldrich), rabbit α H3 (1:10000, ab1791, Abcam) and mouse α tubulin AA4.3 (1:1000; DSHB). Secondary polyclonal Goat antibodies (Sigma) coupled to horseradish peroxidase Rabbit IgG HRP Linked Whole Ab, (NA934) and Mouse IgG HRP Linked Whole Ab (GE Healthcare, NA931) were used at 1:10000. HeLa cells were boiled in 2× Laemmli sample buffer and whole cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes using a Trans Blot System (BioRad). Antibodies used were SPT6 (Abcam, ab32820 rabbit polyclonal; 1:500), CENP-C (Gift from D. Cleveland UCSD, made in house from Covance clone #3024, rabbit polyclonal; 1:10000) and α-tubulin (Sigma, T9026 mouse monoclonal; 1:1000). Secondary antibodies used were Donkey anti-Rabbit 800 (Li-Cor, 926–32211) and Donkey anti-mouse 680 (Rockland, 610-744-124; both at 1:10000). Blots were visualized using Image Lab 6.1 (BIO RAD).
4
Western Blot Analysis of Protein Lysates
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Cells were lysed in RIPA lysis buffer (R0278, Sigma) supplemented with a protease inhibtor (30,496,700, Roche) and phosphatase inhibitor cocktail (P5726, Sigma). Lysates were boiled in 1 × sample buffer (#1610747, Bio-Rad), resolved on 4–20% Mini Protean TGX Precast Gel (#4561096, Bio-Rad), and transferred onto PVDF membranes (IPFL00010, Millipore) using a Bio-Rad Trans-Blot system. Membranes were blocked with 5% BSA in TBS with Tween-20 for 60 min at RT. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. HRP-conjugated secondary antibodies (Bio-Rad Laboratories or Abcam) diluted in blocking buffer at 1:2,500 were incubated for 2h at RT and developed using ECL (WBKLS0100, Miliipore). Blots were imaged using an ChemiDoc MP Imaging System (Bio-rad) and quantified using ImageJ software.
5
Affinity Purification of Protein Complexes
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HEK293T cells were seeded in 10-cm2 dishes (5 × 106) and transfected with the indicated plasmids. If necessary, the total amount of plasmid DNA was supplemented with pcDNA-eGFP. All transfections were performed using TransIT-LT1 (Mirus) following the manufacturer’s instructions. After 2 to 3 days, cells were harvested and lysed in Triton lysis buffer (1% Triton X-100, 20 mM Tris-HCl, 2.5 mM MgCl2). Clarified supernatants were incubated with anti-HA, anti-FLAG, or anti-V5 magnetic beads (1.5 h at 4°C) and washed with high-salt Triton lysis buffer (1% Triton X-100, 20 mM Tris-HCl, 2.5 mM MgCl2, 500 mM NaCl). Proteins were eluted from the beads by adding 2× Laemmli sample buffer and heating the sample at 50°C for 10 min. Proteins were subsequently separated on 4% to 12% Bis-Tris SDS-PAGE gels and transferred to nitrocellulose membranes using a transblot system (Bio-Rad).
6
Protein Expression Analysis via Western Blot
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Total proteins from U20S in different groups were separated by 12% SDS-PAGE, and the separated proteins were next electro-transferred onto nitrocellulose membranes using a transblot system (Bio-Rad, Hercules, CA, USA). The membranes were first blocked with 5% non-fat milk for 2 h at room temperature, followed by incubating with indicated primary Polyclonal rabbit antibody to PI3K (CST, USA, 1:500), p-Akt (CST, USA, 1:500), Akt (CST, USA, 1:1000), PARP (CST,, USA, 1:500), Caspase3 (CST, USA, 1:500), Bax (CST, USA, 1:400), Bcl-2 (CST, USA, 1:1000), β-actin (Abmart, China,1:300) were used as a primary antibody. Anti-rabbit antibody conjugated to HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. β-actin was used as an intrinsic quality control. The bands were incubated in ECL-Plus reagent (Amersham, Piscataway, NJ) and chemiluminescence was detected on BioMax MR Film (Kodak, Rochester, NY). The density of the bands was quantified using a Labworks image acquisition and analysis software (UVP, USA).
7
Western Blot Protein Analysis
8
Western Blotting Detection of Protein Samples
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Protein samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gels electrophoresis (SDS-PAGE, 10%) under reducing conditions [22 (link)]. For Western blotting, samples (30–40 μg of protein) were separated by SDS-PAGE and electro-blotted (35 min, 45 V) in a Bio-Rad transblot system onto nitrocellulose membrane (0.2 μm, Bio-Rad, Hercules, CA, USA) for 90 min. The transfer buffer contained 48 mM Tris, 39 mM glycine, 0.0375% (w/v) SDS and 20% (v/v) methanol. The nitrocellulose was saturated with 5% (w/v) non-fat powdered milk in TBS-T, washed three times with TBS-T, and incubated overnight with 1:200 dilution rabbit anti-Tc/NICT-1 sera. The sheets were washed three times with TBS-T, and immunoreactive proteins were localized using HRP-anti-rabIgG (1:5000) for 1 h at 25 °C. Proteins were detected by chemiluminescence using the SuperSignal West Pico substrate kit (Thermo Fisher, Waltham, MA, USA). Revealed bands were quantified by densitometry using Image J (http://rsbweb.nih.gov , accessed on 21 september 2020).
9
Western Blot Protein Detection Protocol
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Proteins were separated by electrophoresis on 8% or 10% 29:1 polyacrylamide gels in 1× TGS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and transferred onto nitrocellulose membranes using the Bio-Rad TransBlot system. Membranes were saturated by 30 min incubation in TBS-T buffer (Tris-buffered saline with 0.1% Tween 20), containing 5% (w/v) powder milk. Specific proteins were detected by incubation in TBS-T with the following antibodies: anti-HA-HRP (Clone 3F10, Roche) diluted 1000-fold; anti-Dbp6 polyclonal antibodies diluted 10 000-fold (36 (link)); anti-PGK1 (clone 22C5D8, Invitrogen) diluted 10 000-fold; rabbit peroxidase anti-peroxidase soluble complex (PAP, Sigma) diluted 10 000-fold. After incubation with primary antibodies, membranes were washed three times with TBS-T. Anti-rabbit or mouse IgG–HRP conjugate (Promega) were used, when needed, as secondary antibodies. After three washes of the membranes with TBS-T, proteins associated with antibodies were detected as chemiluminescent signals using Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc imager apparatus (Bio-Rad) followed by quantification with the ImageLab software (Bio-Rad).
10
Deubiquitinase Activity Profiling of ISG15
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OTU activity towards cellular conjugated ISG15 was determined using purified OTUs and cell lysates. Cell lysates containing conjugated human ISG15 were obtained by treating Huh7 cells with 1000 IU of IFN-β for two days before harvesting in triton lysis buffer (1% triton X-100, 20 mM Tris-HCl, 2.5 mM MgCl2). Alternatively, lysates were obtained by transfecting HEK293T cells with plasmids expressing V5-tagged sheep or human ISG15 (Genscript), and human Ube1L, UbecH8, and HERC5 using Trans-IT-LT1 (Mirus). Lysates were clarified by centrifugation (20 min at 13,000 rpm) and incubated for 20 min at room temperature with purified OTU. Samples were mixed with 4x Laemmli buffer and boiled at 95°C for 8 minutes. Proteins were separated on 4–12% Bis-Tris SDS-PAGE gels and transferred to nitrocellulose membranes using a trans-blot system (BioRad). ISG15 was detected using an ISG15 antibody (Proteintech # 15981-1-AP) or a V5 tag antibody (ThermoFisher Scientific #R960CUS). Tubulin was used as loading control marker (T5169, Sigma). Primary antibodies were detected with SuperSignal West Dura Fast Western blot kits (Thermo Fisher). Protein bands were visualized using a ChemiDoc MP system (BioRad).
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