Protein lysates were prepared as previously described [11 ]. Briefly, 50 mg of the liver was homogenized in 1 mL of cell lysis buffer, 1X proteinase inhibitor, and 1X PMSF. Samples were sonicated on power 4, thrice for 5 s each, and then, spun at 13,200 RPMs for 15 min at 4 °C. Protein concentrations were determined using a BCA reagent (23,227; Pierce™). Protein lysates were resolved in 3–8% Tris-Acetate Novex gel (Invitrogen) for NCOR1, SMRT/NCOR2, and beta-actin or 10% Bis-Tris gel (Invitrogen) for hdac3 and hsp90, and then, transferred to a nitrocellulose membrane. Blots were probed for NCOR1 using rabbit polyclonal anti-NCOR1 antibody (PHQQ; generated against a C-terminal NCoR peptide as previously described; diluted at 1:250), rabbit polyclonal anti-SMRTe (06–891; Millipore Sigma; diluted at 1:500), or rabbit polyclonal anti-HDAC3 (ab7030; abcam; diluted at 1:2000) overnight at 4 °C with mouse monoclonal anti-beta-actin antibody (MA191399; Fisher Scientific; diluted at 1:10,000) for NCOR1 and SMRT/NCOR2 control or mouse monoclonal anti-hsp90a/b antibody (sc-13119, Santa Cruz Biotechnology; 1:5,000). Goat anti-rabbit Alexa Fluor Plus 680 (A32734; Invitrogen) and goat anti-mouse Alexa Fluor Plus 488 (A32723; Invitrogen) were used as secondary antibodies and incubated at room temperature for 90 min. Images were captured on ChemiDoc Touch MP (Bio-rad).
Chemidoc touch mp
Manufactured by Bio-Rad
Sourced in United States, Japan
The ChemiDoc Touch MP is a high-performance imaging system designed for versatile imaging applications in the laboratory. It captures images of various biological samples, such as Western blots, gels, and membranes, using sensitive detection technology. The system provides a touchscreen interface for intuitive and efficient image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Ab98425, by Abcam (2 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab7030, by Abcam (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Sc 13119, by Santa Cruz Biotechnology (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Precision plus protein all blue prestained protein standard, by Bio-Rad (1 mentions)
Quick cbb, by Fujifilm (1 mentions)
Powerpac 3000, by Bio-Rad (1 mentions)
Supersep tm ace gel, by Fujifilm (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Phospho kinase array, by R&D Systems (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence plus reagent, by GE Healthcare (1 mentions)
20 protocols using chemidoc touch mp
1
Protein Lysate Preparation and Western Blotting
2
Immunoblotting of Cellular Proteins
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma #P8340). After measurements of concentration using DC protein assay kit (BioRad #500112), the same amount of proteins were run on SDS-PAGE gel, followed by transfer onto nitrocellulose membrane. The membrane was incubated with antibody for LC3 (1:1000), acetyl-Histone H3 (Lys9) (1:1000), Histone H3 (1:1000), p21 (1:1000), or β-actin (1:2000) at 4 °C for overnight, following blocking with 5% skim milk. After washing with TBS-T, the membrane was incubated with HRP-labelled appropriate secondary antibody (1:2000), followed by signal detection using ChemiDoc Touch MP (BioRad). Results were confirmed by at least two independent experiments.
3
Non-Reducing SDS-PAGE Analysis of BsAb
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BsAb samples were subjected to non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether the disulfide bonds between HC and LC were correctly formed. Each 1 μg sample was mixed with SDS Sample Buffer Solution (2ME-) (× 4) (Wako) and heated at 95 °C for 2 min, and then loaded onto a Mini-PROTEAN TGX gel (4–20%, 15 well) (Bio-Rad) alongside a molecular weight marker (Precision Plus Protein™ All Blue Prestained Protein Standards, Bio-Rad). Electrophoresis was performed using a Power PAC 3000 (Bio-Rad) at a constant voltage of 200 V for 35 min. The gel was then removed from the cassette and stained with Quick-CBB (Wako), followed by destaining in Milli-Q water. The stained gel was imaged using a ChemiDoc Touch MP (BIO-Rad).
4
Western Blot Analysis of Exosomal Markers
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Cells were lysed in ice-cold lysis buffer (1% NP-40, 50 mM Tris (pH 7.5), 165 mM NaCl, 10 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin). Proteins were separated on 10% (CD63 and AMIGO2) or 12% (CD9) sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (ISEQ00010; Merck Millipore, Darmstadt, Germany). The membranes were blocked using 2.5% skim milk (prepared in T-PBS) at room temperature (RT) for 2 h. The membranes were then probed with the following primary antibodies: rat monoclonal anti-AMIGO2 antibody (1:10,000; rTNK1b012a)33 , mouse monoclonal anti-β-actin antibody (1:5,000; A5441; Sigma Aldrich, St. Louis, MO, USA), mouse monoclonal anti-CD9 antibody (1:2,500; 12A12; Cosmo Bio, Tokyo, Japan), or mouse monoclonal anti-CD63 antibody (1:5,000; 8A12; Cosmo Bio) overnight at 4 °C. The membranes were then incubated with a goat polyclonal anti-rat IgG horseradish peroxidase-conjugated antibody (1:10,000; ab98425; Abcam, Cambridge, United Kingdom) or a goat polyclonal anti-mouse IgG antibody (1:5,000; PM009-7; MBL, Nagoya, Japan) at RT for 20 min. Protein signals were detected using an enhanced chemiluminescence kit (RPN2232; GE Healthcare; Buckinghamshire, United Kingdom) and analyzed using ChemiDoc Touch MP (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of Denatured Proteins
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Lysate was mixed with 43 SDS Sample Buffer and beta-mercaptoethanol, and proteins were denatured by boiling for 5 min. Denatured proteins were separated on a 5–20% SuperSep TM Ace gel (Wako) in 25 mM Tris/192 mM Glycine/0.1% SDS Running Buffer and transferred to a PVDF membrane (Merck Millipore) in 25 mM Tris/192mM Glycine/20% Methanol Transfer Buffer with Mini Trans-Blot Cell (Bio-Rad). Blots were blocked with 5% skim milk in TBS-T for 30 min, and incubated with primary antibodies (which are listed in the key resource table ) at 4°C overnight. Immunocomplexes were labeled by HRP-conjugated anti-mouse IgG or anti-rabbit IgG and visualized using Amersham ECL Prime (Cytiva). The signals were deteceted with ChemiDoc Touch MP (Bio-Rad).
6
GPER-1 siRNA Phosphokinase Profiling
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After transfection of GPER‐1 siRNA, the protein expression profiles were examined using the Phospho‐Kinase Array (ARY003B®, R&D Systems Inc.). Phosphokinase signals were detected using X‐ray films following exposure to chemiluminescent reagents. The array was visualized using a ChemiDoc Touch MP (Bio‐Rad Laboratories Inc.). We quantified protein phosphorylation levels using a pixel density module in ImageJ.
7
Protein Expression Analysis of p21 and GAPDH
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma #P8340). After measurements using DC protein assay kit (BioRad #500112), the same amount of proteins were run on SDS-PAGE gel, followed by transfer onto nitrocellulose membrane. The membrane was incubated with p21 (1:1000), or GAPDH (1:3000) antibody at 4 °C for overnight, following blocking with 10% skim milk. After washing with TBS-T, the membrane was incubated with HRP-labelled secondary antibody (1:2000), followed by detection using ChemiDoc Touch MP (BioRad).
8
Spinal Cord Injury Histone and Cytokine Analysis
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The motor cortex and the lesion site of the spinal cord tissues were collected on the indicated days after SCI. Tissue lysates were prepared in NP-40 lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 10 mM NaF, 1 mM Na3VO4) containing a protease inhibitor cocktail (Roche Diagnostics) at 4 °C, subjected to SDS-PAGE, and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocked with 5% Skim milk in the PBS-T (0.05% Tween-20) for 60 min, membranes were incubated with anti-acetylated histone H3 or anti-histone H3 antibody at 4 °C overnight. Enhanced chemiluminescence plus reagents (GE Healthcare) were used for detection. For cytokine array, mouse cytokine array panel A (R&D Systems, Minneapolis, MN, USA) was used. The spinal cord tissues were prepared 3 days after SCI, and tissue lysates were incubated with a cytokine array membrane according to manufacturer’s protocol. The signals were analyzed using an Amersham imager system (GE Healthcare) or ChemiDoc Touch MP (Bio-Rad, Richmond, CA, USA). The signal intensity was measured using the Image Lab software (Bio-Rad).
9
Fluorescent Oligonucleotide Degradation Assay
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Various oligonucleotides (40 pmol), of which AS and CS were labeled with AF647 and AF488, respectively, were incubated with 1 ng of RNase A (QIAGEN, Hilden, Germany) or 10 units of RNase H (Takara Bio) at 37°C for 1 h and then loaded on 20% polyacrylamide gel. After electrophoresis in TBE, fluorescence of each dye was imaged using ChemiDoc Touch MP (Bio-Rad, Hercules, CA, USA).
10
Characterization of RBD-siRNA Interactions
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Cy3-labelled siRNA (100 nM) was mixed with 0–800 nM purified RBD fusion protein in binding buffer [20 mM Tris–HCl (pH 7.6), 100 mM NaCl, 1 mM EDTA, 0.02% (v/v) TritonX-100, 2 mM dithiothreitol] at 4 °C for 1 h. The samples were then analysed by electrophoresis, where 5 × TBE sample buffer [90 mM Tris–HCl (pH 7.6), 90 mM boric acid, 2 mM EDTA, 15% Ficoll type 400, 0.02% xylene cyanol] was diluted to 1 × TBE in the binding reaction, and then 12.5 μL of the sample was applied to a 6% TBE gel and electrophoresed at a constant voltage of 100 V for 60 min in 0.5 × TBE buffer. Cy3 fluorescence was detected using ChemiDoc Touch MP (BioRad).
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