Protein was extracted using RIPA lysis buffer (EMD Millipore Billerica, MA, USA, 10 × RIPA buffer) and quantified via the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). The protein samples were mixed with SDS loading buffer and separated on an 8–12% SDS-PAGE gel and transferred to PVDF membrane (NEF1002001PK, Perkin Elmer, Waltham, MA, USA). The membrane was then blocked with 5% BSA (Sigma) at room temperature for 1 h in Tris-buffered saline-Tween 20 (0.5%; TBS-T). After washing in TBS-T, membranes were incubated overnight at 4 °C in 2.5% BSA with the appropriate primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. All antibodies and dilutions are shown in Table S2. The immunoblot signals were developed using the Super Signal Ultra chemiluminescent reagent (Pierce, Rockford, US) and detected by a CCD camera (MultiGel-21, Topbio, Taipei, Taiwan).
Multigel 21
Manufactured by Top-Bio
Sourced in United States
MultiGel-21 is a multi-channel gel electrophoresis system designed for the simultaneous separation of multiple samples. It features a modular design and accommodates a range of gel sizes to support various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus software, by Media Cybernetics (3 mentions)
Ecl detection system, by Merck Group (3 mentions)
Bradford method, by Bio-Rad (3 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Nef1002001pk, by PerkinElmer (1 mentions)
Crl1729, by American Type Culture Collection (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Peroxidase labeled goat anti mouse secondary antibodies, by Cytiva (1 mentions)
Peroxidase labeled goat antirabbit secondary antibodies, by Cytiva (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Rad51, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
P21 waf1 cip1, by Cell Signaling Technology (1 mentions)
18 protocols using multigel 21
1
Western Blot Protein Detection Protocol
2
Analyzing Histone Acetylation and Cell Signaling in Cancer Cells
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Cells were seeded at a density of 1 × 106/10 cm dish. Following treatment with BMX (0, 5, and 10 μM), and suberoylanilide hydroxamic acid (SAHA), Valproic acid (VPA) or PCI-34051 in the presence or absence of TMZ (50 μM) or Oxp (5 μM) for 48 h, the lysates were analyzed by Western blotting as described previously [8 (link)]. The specific primary antibodies against acetyl-histone H3 (Lys9/Lys14), acetyl-histone H4 (Lys8), p53, acetyl-p53 (Lys382), phospho-p53 (Ser15), p21, p16, MGMT, phosphor-γ-H2AX (Ser139), E2F1, E2F3, cleaved caspase 3, cleaved caspase 8, cleaved caspase 7, cleaved caspase 9, PARP, Bax, Bcl-2, Bid, Bim, Bak, Puma, β-catenin, phospho-β-catenin (Ser/33/37/41), GSK3β, phospho-GSK3β (Ser 9), c-Myc, Cyclin D1, p62, LC3I/II, CD133, CD44, SOX-2, and HDAC8 were used for detection, and GAPDH, α-tubulin or β-actin was used as the internal control. After incubation with the primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, the intensities were developed using a chemiluminescent solution (Pierce, Rockford, US) and detected using a gel imager CCD camera (MultiGel-21, Topbio, Taipei, Taiwan). All antibodies and their dilutions are shown in Additional file 1 : Table S1.
3
Yeast Protein Detection and Quantification
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All Saccharomyces cerevisiae strains used in this study are listed in Table 1 . Unless otherwise indicated, all strains were grown at 30 °C in rich medium (yeast extract peptone) or synthetic dropout medium containing 2% glucose (Glc). The plasmids used in this study are listed in Table 2 .
Anti-Loc1, anti-Puf6, anti-Tif6, anti-Rpl23, anti-Rpl8 [38 (link)], and anti-Rpl43 antibodies were generated in the lab. Anti-myc antibody was obtained from MYC 1-9E10.2 [9E10] (ATCC® CRL1729™). Anti-HA (hemagglutinin) (MMS-101P), anti-TAP (Tandem affinity purification) (CAB1001), and anti-GST (glutathione S-transferase) (GST001M) were purchased from Covance, Thermo, and Bioman, respectively. Proteins were resolved in SDS-PAGE and transferred to a membrane with Trans-Blot® SD Semi-Dry (BioRad). Signals were detected using ClarityTM Western ECL substrate (Biorad, Hercules, CA, USA) and scanned by MultiGel-21 (Top Bio, Taipei, Taiwan). The signal intensity was by measured by Image J for quantification.
Anti-Loc1, anti-Puf6, anti-Tif6, anti-Rpl23, anti-Rpl8 [38 (link)], and anti-Rpl43 antibodies were generated in the lab. Anti-myc antibody was obtained from MYC 1-9E10.2 [9E10] (ATCC® CRL1729™). Anti-HA (hemagglutinin) (MMS-101P), anti-TAP (Tandem affinity purification) (CAB1001), and anti-GST (glutathione S-transferase) (GST001M) were purchased from Covance, Thermo, and Bioman, respectively. Proteins were resolved in SDS-PAGE and transferred to a membrane with Trans-Blot® SD Semi-Dry (BioRad). Signals were detected using ClarityTM Western ECL substrate (Biorad, Hercules, CA, USA) and scanned by MultiGel-21 (Top Bio, Taipei, Taiwan). The signal intensity was by measured by Image J for quantification.
4
Embryonic Bad Protein Analysis
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The embryos were injected with bad MO and control MO at the one-cell stage using a gas-driven microinjector (Medical System Corporation, Greenvale, NY, USA). Fifty embryos were collected at 24 hpf and homogenized in lysis buffer (10 mM Tris-HCl, pH 6.8, 20% glycerol, 10 mM sodium dodecyl sulfate (SDS), and 2% β-mercaptoethanol). An aliquot of each lysate with 40 μg protein per sample was separated by electrophoresis on an SDS polyacrylamide gel to resolve the proteins. The gels were immunoblotted with the following antibodies: anti-bad antibodies (BD Transduction Laboratories) and anti-β-actin monoclonal antibodies (BD Pharmingen), followed by peroxidase-labeled goat anti-mouse secondary antibodies (1:15,000 dilution; Amersham Biosciences, Piscataway, NJ, USA) or peroxidase-labeled goat antirabbit secondary antibodies (1:7500 dilution; Amersham Biosciences, Piscataway, NJ, USA). Chemiluminescence indicative of antibody binding was captured by MultiGel-21 (TOP BIO, Taipei, Taiwan) as previously described [55 (link)].
5
Profiling Histone Deacetylase Signaling
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Proteins were extracted with RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA), prepared in 5X sodium dodecyl sulfate (SDS) buffer, and analysed by SDS–PAGE (10% polyacrylamide gel). Following transfer to the polyvinylidene difluoride (PVDF) membrane, antibodies targeting HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, acetyl-histone 3 (Lys9), acetyl-α-tubulin (Lys40), phospho-PTEN (Ser380/Thr382/383), PTEN, phospho-Akt (Ser473), Akt, Rad51, p21 Waf1/Cip1, Snail and GAPDH (Cell Signaling Technology, Danvers, MA) were used for immunoblotting (Supplementary Table S1 ). GAPDH was detected as the internal control. Protein bands were detected with an enhanced chemiluminescence (ECL) kit (Amersham International plc, Buckinghamshire, England) and analysed by MultiGel-21 (Topbio, Taiwan).
6
Protein Expression Analysis in ARPE-19 Cells
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ARPE-19 cells in each group were removed from the supernatant and lysed with cell lysis buffer (containing 10 mM Tris, 0.1% Triton X-100, and 1 mM EDTA) to collect protein samples. To analyze the expression levels of specific proteins, all samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes for binding with corresponding antibodies. After adding ECL substrate, all proteins were visualized using MultiGel-21 (GIS-21-C2-6M, TOPBIO) for imaging (Taipei, Taiwan). The antibodies, including cleaved caspase-3 (31A106), p-MEK-1/2 (7E10), p-ERK (12D4), p-p38 (D8), p-JNK (G7), and Fis1 (B5) were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cleaved caspase-9 (9505T), cleaved PARP (Asp214, D64E10), and p-Drp-1 (3455S) were purchased from Cell Signaling Technology (Beverly, MA, USA).
7
Protein Extraction and Western Blot Analysis
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Total cell lysate extraction and protein contents were respectively prepared and determined as previously described [25 (link)]. Equal amounts of protein extracts (20~30 μg) were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and probed with indicated primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. After washing, blots were incubated with enhanced chemiluminescence reagent (Amersham, Arlington Heights, IL) and signals were detected by the chemiluminescence imaging system, MultiGel-21, (TOP BIO, New Taipei City, Taiwan). Densitometric analysis of the bands was carried out using Image-Pro Plus software (Media Cybernetics, Rockville, MD).
8
Western Blot Analysis of Protein Samples
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Protein samples (20–50 μg per lane) were separated through 7–13% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrotransferred onto BioTrace NT Membranes (Pall, Port Washington, NY, USA). These membranes were then incubated with primary and secondary antibodies at optimum dilutions, and immunoreactive signals were detected using an Immobilon Crescendo Western HRP substrate (Millipore Burlington, MA, USA) and MultiGel-21 (TOPBIO, New Taipei City, Taiwan). The antibodies used and their dilutions are listed in Additional file 1 : Table S1.
9
Amiodarone-Induced Autophagy in Liver Cells
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HepG2 and CRL 1439 cells cultured with or without amiodarone were harvested and total cell protein was extracted using whole cell lysis buffer. The protein concentrations were determined by the Bradford method (Bio-Rad, Berkeley, CA, USA). Samples with equal amounts of protein were subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidenedifluoride (PVDF) (Millipore, Bedford, MA, USA) membrane. The membrane was incubated at room temperature in blocking solution (5% nonfat milk) for 1 h followed by incubation for 2 h in blocking solution containing an appropriate dilution of anti-LC3B, -P62, -Beclin-1 antibody (Cell Signaling Technology, Danvers, Massachusetts, USA). After washing, blots were then probed with appropriate secondary horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), detected by an ECL detection system (Millipore, Burlington, Massachusetts, USA), and scanned by MultiGel-21 (Top Bio, Taipei, Taiwan). β-actin was indicated as an internal control.
10
Western Blot Analysis of Protein Lysates
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Protein lysates were prepared as described previously30 (link). The protein content was determined with the Bio-Rad protein assay reagent using bovine serum albumin as a standard. A western blot analysis was performed with indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. After washing, blots were incubated with the western blotting reagent ECL, and chemiluminescence was detected by the chemiluminescence imaging system, MultiGel-21, (TOP BIO, New Taipei City, Taiwan). Furthermore, the same blots were stripped with stripping buffer (TOOLS, New Taipei City, Taiwan) and reprobed with a β-actin or α-tubulin antibody as the internal control. Densitometric analysis of the bands was carried out using Image-Pro Plus software (Media Cybernetics Inc. Rockville, MD) to define the boundaries of protein bands, assess density, and to obtain final numerical data. The intensity of bands from the protein of interest was normalized to the intensity of actin or tubulin bands of the respective blots.
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