At 48 h post transfection of 5×105 SH-SY5Y cells, total protein was extracted. Equal amounts (15 μg protein/lane) of proteins obtained from whole cell extracts of untransfected and Elk1 transfected samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane by Trans-Blot Turbo Blotting System (Bio-Rad). Membrane was blocked in 5% skim milk powder/TBST (Tris buffered saline (TBS) containing 0.1% Tween 20) for 1 h at room temperature, and then incubated with the following primary antibodies at indicated dilutions overnight at 4°C; rabbit monoclonal His-tag antibody (1:1000, Cell Signaling Technology), mouse monoclonal spastin antibody (1:1000, Sigma), rabbit polyclonal katanin-p60 antibody (1:1000, ATLAS), mouse monoclonal p27 antibody (1:500, Santa Cruz), mouse monoclonal HuR antibody (1:500, Santa Cruz), mouse monoclonal β-Actin antibody (1:1000, Cell Signaling Technology), rabbit monoclonal GAPDH antibody (1:1000, Cell Signaling Technology), rabbit monoclonal β-tubulin antibody (1:1000, Cell Signaling Technology) in 5% skim milk powder/TBST. Membranes were then incubated with Horseradish peroxidase conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (1:3000, Cell Signaling Technology) for 1 h at room temperature. Bands were visualized using Visualizer Western Blot Detection Kit (Millipore) and ChemiDoc Imaging System (Bio-Rad).
Visualizer western blot detection kit
Manufactured by Merck Group
Sourced in United States
The Visualizer Western Blot Detection Kit is a laboratory equipment product designed for the detection and visualization of proteins in Western blot analysis. It provides the necessary reagents and components to facilitate the chemiluminescent detection of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Iblot, by Thermo Fisher Scientific (2 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Protein loading buffer, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Las 4000 imager, by Fujifilm (2 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (2 mentions)
Nonfat milk powder, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg secondary antibodies, by Cell Signaling Technology (1 mentions)
Mouse monoclonal β actin antibody, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
P raf, by Abcam (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
Hnf4α, by Abcam (1 mentions)
9 protocols using visualizer western blot detection kit
1
Western Blot Analysis of Elk1 Overexpression
2
Quantitative Protein Expression Analysis
3
Western Blot Quantification of EMT Markers
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The operation of the Western blot was referenced the previous instructions [23 (link)]. Briefly, the protein was combined with primary antibodies, and the corresponding secondary antibody was applied to bind to the unique antibody. In the end point, the special protein was visualized via a Visualizer Western Blot Detection Kit (Millipore, Bedford, MA, U.S.A.). The primary antibodies were introduced as follows: HNF4α (ab201460, 1:1000), E-cadherin (ab76055, 1:1000), Vimentin (ab8979, 1:1000), N-cadherin (ab18203, 1:500), phospho-RAF (p-RAF; ab173539, 1:3000), GAPDH (ab8245, 1:5000), RAS (3965, 1:1000), phospho-ERK (p-ERK; 9101, 1:1000), and phospho-MEK (p-MEK; 9154; 1:1000). Of which HNF4α, E-cadherin, Vimentin, N-cadherin, p-RAF, and GAPDH were purchased from Abcam (Cambridge, MA, U.S.A.), RAS, p-ERK, and p-MEK were obtained from Cell Signaling Technology (Boston, MA, U.S.A.).
4
Protein Extraction from Testes and Sperm
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To prepare the proteins from testes and mouse/human sperm, the samples were lysed in Radioimmunoprecipitation assay buffer and then homogenized on ice. The lysates were then centrifuged at 12,000 × g for 10 min at 4 °C to collect the supernatant. Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime). All samples were processed using 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 h at room temperature with 5% BSA and then incubated with primary antibodies overnight at 4 °C. After incubation with labeled secondary antibodies, signals were obtained using a Visualizer Western Blot Detection Kit (Millipore). The primary antibodies are listed in Table S1 .
5
Western Blot Analysis of CD44 Protein
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Gene expression was analyzed using western blot analysis. Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein (40 μg) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80 °C for 5 min, electrophoresed on a 4–20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4 °C, incubated with HRP-coupled secondary antibody 1 h at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1: 1000 dilutions: human anti-CD44 (#3570S, Cell Signaling Technology) and mouse anti-actin (MAB 1501R, Millipore). Secondary antibody, anti-mouse-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:10,000 dilutions.
6
Western Blot Protein Detection
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Western blot was performed for detection of specific proteins in our cell culture experiments. The protocol started with uploading 30 μg proteins from the whole cell lysate in each sample onto a 10% PAGE gel. After electrophoresis and gel transferring, the membrane was blocked with 5% non-fat milk in 1xTris-buffered saline (pH 7.4) containing 0.05% Tween-20, and then probed with primary antibodies at concentrations of 1:1000 for β-actin (Santa Cruz Biotechnology, US), 1:2000 for CHEK1 and Cyclin E and 1:10000 Cyclin D1 (both from Epitomics, US). Secondary antibodies were added at concentrations of 1:10,000 to 1:20,000. The detected proteins were visualized using the Visualizer Western Blot Detection Kit (Millipore, US).
7
Protein Expression Analysis in GC Cells
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Expression levels of AR, PARP, cleaved PARP, BAX, BCL-2, caspase-6, Flag (DDDDK-tag), BIK, Ki-67, and β-actin proteins in GC cell lines or GC tissues were detected by Western blot analysis according to the protocol of our previous studies. Protein was extracted from tissues and cells at 24 or 48 h post-transfection using RIPA buffer. The protein concentration was measured by using a BCA protein assay kit (Thermo Scientific, CA, USA). Approximately 40 μg of lysates per sample were analyzed using standard western blotting assay procedure. Briefly, the lysates were separated by SDS–PAGE using 10% polyacrylamide gels and transferred onto a PVDF membrane for 1.5 h. The membrane was blocked with 5% skim milk followed by incubation with the primary antibodies summarized in Supplementary Table S7 at 4 °C overnight. After 3 × 10 min washes with TBST at room temperature, the membrane was incubated with HRP-conjugated secondary antibody Mouse Anti-Human (1:2000) (Santa Cruz, USA) for 1 h at room temperature. The detected proteins were visualized using the Visualizer Western Blot Detection Kit (Millipore, USA). Detection was performed by C-DiGit Chemiluminescent Western Blot Scanner (LI-COR, USA).
8
Western Blot Analysis of Cell Signaling
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Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein(50ug) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80°C for 5 minutes, electrophoresed on a 4-20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4°C, incubated with HRP-coupled secondary antibody 1 hour at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1:1000 dilutions: Rabbit anti-FOXM1 (#5436), PLK1(#4513), pCDC2(#9111), CDC2 (#9112), MRE11, Survivin (#4895), mouse anti-STAT3(#9139), pSTAT3 (#9138), ß-Actin (#3700) (Cell Signaling Technology., MA); mouse-anti -pγH2AX (#05636, Millipore., MA), rabbit-anti RAD51 (sc-8348, Santacruz., CA), mouse anti-53BP1 (#612522, BD Transduction Laboratories., CA). Secondary antibodies, goat anti-rabbit-HRP, goat anti-mouse-HRP (Santa Cruz, CA) were used at 1: 10,000 dilution.
9
Quantitative Western Blot Analysis of EV Proteins
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Cells, 10K and 100K pellets were lysed in Pierce RIPA buffer (25mM Tris-HCl (pH 7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktail (Roche) on ice for 20 mins. Cell pellets were spun down at 13,000g for 15 mins at 4 o C. Protein concentration was determined using the DC Protein Assay (BioRad). For whole cell lysates, 30 µg protein extracts were resolved using 4-12% SDS-PAGE gels (Life Technologies) and transferred to nitrocellulose membranes using iBlot (Life Technologies). For 10K and 100K fractions, the same number of cells were seeded at the beginning of each experiment, and EV pellets were resuspended in the same volume of RIPA buffer and same volume loaded for SDS-PAGE. Membranes were probed with primary antibodies overnight on a 4°C shaker, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and signals were visualized with Visualizer™ Western Blot Detection Kit (Millipore). Band analysis was performed using ImageLab software from BioRAD and normalized to cell counts.
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