Samples of cell-free liver extract containing 50 μg of proteins were heated for 5 min in Laemmli buffer (Sigma, St Louis, MO, United States), separated by SDS PAGE using 15% gel in a Mini trans-blot transfer cell, and then transferred to nitrocellulose membrane using a mini trans-blot (Bio-Rad Laboratories, Hercules, CA, United States). The membrane was blocked with TBS-T buffer containing 5% non-fat dry milk. For immunodetection, the blots were incubated overnight at 4°C with monoclonal mouse anti-MT antibody (Enzo Life Sciences) diluted in TBS-T buffer containing 5% BSA. The blots were then washed and incubated with a peroxidase linked secondary antibody (Santa Cruz). Protein loading was verified using anti-β-actin antibody (Santa Cruz). Immunodetection was performed by using an enhanced chemiluminescence kit, and autoradiographs were obtained by exposure to X-ray Films. Immunoblots were digitalized, and the densitometric analysis of the bands obtained was carried out using WCIF ImageJ based on 256 grey values (0 = white; 256 = black). Quantification of the bands was obtained by measuring (5 times on each band) the mean optical density of a square area after the background area was subtracted. The results of absorbance measurements and the grey values obtained from the densitometric analysis were expressed as mean ± SE of five determinations for each sample.
Mini trans blot
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom
The Mini Trans-Blot is a compact, self-contained semi-dry electroblotting system for the efficient transfer of proteins from polyacrylamide gels to membranes. It features a simple, easy-to-use design and provides a rapid and reliable method for protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Las 4000, by Cytiva (5 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Hybond, by Cytiva (4 mentions)
Immobilon p, by Merck Group (4 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Luminata forte western hrp substrate, by Merck Group (3 mentions)
Immobilon, by Merck Group (2 mentions)
Gs 800 densitometer, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Whatman, by Cytiva (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Mini protean tetra, by Bio-Rad (2 mentions)
Molecular analyst, by Bio-Rad (2 mentions)
Ripa containing protease and phosphatase inhibitors, by Roche (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Amersham ecl prime, by Cytiva (2 mentions)
Western max hrp chromogenic detection kit, by Avantor (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Peroxidase linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
84 protocols using mini trans blot
1
Western Blot Analysis of Metallothionein
2
Western Blot Analysis of Akt Pathway
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To ensure equal sample loading, protein concentration was determined using bicinchoninic acid (BCA) protein assay reagent (Sigma, UK) and adjusted accordingly. NuPAGE LDS Sample Buffer (4X) (Thermofisher Scientific, UK) plus 5% β-mercaptoethanol were added to the samples, which were subsequently denatured by heating to 100 °C for 10 min. Samples were then loaded on NuPAGE Novex 10% Bis–Tris protein gels (Thermofisher scientific, UK) using the Mini Protean III system (Bio-Rad, UK). Proteins were transferred onto nitrocellulose blotting membrane (GE Healthcare Life Sciences, UK) through wet transfer in a Bio-Rad Mini Trans-Blot. The membranes were blocked for 1 h using 5% bovine serum albumin/PBS tween and subsequently incubated with appropriate primary antibodies at 4 °C overnight.
The following primary antibodies being used were acquired from Cell Signalling Technology: Akt (#9272), Phospho-Akt (Ser473) (#9271), Phospho-Akt (Thr308) (#2965), and PI3 Kinase p110α (#4249). Anti-GAPDH (mAbcam, #9484) was used as loading control. After overnight incubation, membranes were probed with secondary antibodies. Levels of protein were finally quantified using the Odyssey imaging system from Li-Cor Biosciences (Image Studio Lite Ver 5.2).
The following primary antibodies being used were acquired from Cell Signalling Technology: Akt (#9272), Phospho-Akt (Ser473) (#9271), Phospho-Akt (Thr308) (#2965), and PI3 Kinase p110α (#4249). Anti-GAPDH (mAbcam, #9484) was used as loading control. After overnight incubation, membranes were probed with secondary antibodies. Levels of protein were finally quantified using the Odyssey imaging system from Li-Cor Biosciences (Image Studio Lite Ver 5.2).
3
SDS-PAGE Protein Separation and Western Blot
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Electrophoretic separation of proteins was done by a SDS-PAGE. Electrophoresis was performed in a mini gel apparatus (Protean 3, Biorad) at 100 V, before the gel was transferred to a tank-blot-system (Mini Trans blot, Biorad) onto a PVDF membrane (Immobilon, Millipore). After blotting (1 h, 100 V) the membrane was dried and then blocked with 5% milk powder/TBS for 1.5 h. Primary and secondary antibodies and dilutions used are listed below. Incubation with primary antibody was done overnight at 4°C. For detection, a HRP based ECL Kit (ECL system, Amersham) was used. Therefore, the membrane was incubated with HRP conjugated secondary antibody (HRP, Jackson) at RT for 1 h. The exposed films (Hyperfilm ECL, Amersham) were developed by a Curix 60 (Agfa).
4
Western Blot Protocol for His-tagged Protein Detection
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Proteins from SDS-PAGE were transferred electrophoretically to nitrocellulose membrane (Genetix, India) using mini-transblot (Biorad, USA) at 30 V overnight at 4°C
[33 (link)] and the membrane was blocked with 3% BSA (Amresco, USA) in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). For immunodetection of the His fusion proteins, the primary antibody used was His probe (H-3) mouse monoclonal IgG (1 : 500; Santa Cruz Biotechnology, USA) and the secondary antibody was HRP-conjugated anti-mouse IgG antibody (1 : 2000; Santa Cruz Biotechnology, USA). Bound antibodies were detected using di-aminobenzidine (DAB) (Amresco, USA) as substrate.
[33 (link)] and the membrane was blocked with 3% BSA (Amresco, USA) in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). For immunodetection of the His fusion proteins, the primary antibody used was His probe (H-3) mouse monoclonal IgG (1 : 500; Santa Cruz Biotechnology, USA) and the secondary antibody was HRP-conjugated anti-mouse IgG antibody (1 : 2000; Santa Cruz Biotechnology, USA). Bound antibodies were detected using di-aminobenzidine (DAB) (Amresco, USA) as substrate.
5
Investigating Angiogenesis Signaling Pathways
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Rabbit HIF-1α polyclonal antibodies (Upstate Biotechnology, USA); VEGF polyclonal antibodies, albumin-fluorescein isothiocyanate conjugate (FITC-albumin), β-actin monoclonal antibodies (Sigma-Aldrich Corporation, USA); IgG secondary antibodies (Beijing Zhongshan Gold Bridge Biotechnology Corporation Ltd, China); newborn calf serum (Chengdu Harry Biotechnology Corporation Ltd, China); primer synthesis (Sangon Biotechnology (Shanghai) Corporation Limited, China); Roswell Park Memorial Institute (RPMI) 1640 culture medium (HyClone Laboratories, USA); Bicinchoninic acid (BCA) protein assay kit (PierceTM, Thermo Fisher Scientific, USA); Mini Trans-Blot® electro transfer system, fluorescence quantitative PCR instrument, ChemiDoc XRS Gel Analysis System, “Quantity one” software (Bio-Rad Laboratories, USA); MilicellTM electrical resistance system (ERS) instrument (Millipore, USA); inverted fluorescence microscope model TH4-200 (Olympus Corporation, Japan); ultra-sensitive enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA); HeraeusCO2 constant temperature incubator (Heraeus, Germany); DU®-640 ultraviolet spectrophotometer (Beckman Inc., USA); modular incubator chamber (Billups-Rothenberg Inc., USA).
6
Western Blot Analysis of COX-1, COX-2, and PLIN2
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For the analysis of COX-1, COX-2, and PLIN2 protein content, whole cell extracts were obtained by lysing the cell pellets with 80 mL of sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M β-mercaptoethanol, 0.1% bromophenol blue) and then boiled for 10 min at 100°C. Samples were resolved by electrophoresis (SDS-PAGE) on a 10% separation gel overlaid with a 5% stacking gel. After that, proteins were transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using a Mini Trans-Blot® (Bio-Rad Laboratories, Richmond, CA, USA), and the membranes were blocked for 1 h with 5% nonfat dry milk in TTBS (20 mM Tris, 100 mM NaCl and 0.5% Tween 20). Membranes were then incubated with a primary rabbit antibody against COX-1 or COX-2 or PLIN2 (Abcam, San Francisco, USA) overnight at 4°C or with β-actin (Sigma, San Louis, USA) for 1 h at room temperature. They were then washed and incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1 : 1000 dilution, 1 h, at room temperature). Detection was by the enhanced chemiluminescence (ECL) method according to the manufacturer's instructions (GE Healthcare, Buckinghamshire, UK).
Band densities were quantified with a GS 800 Densitometer (Bio-Rad Laboratories, Richmond, CA) using Molecular Analyst® image analysis software (Bio-Rad Laboratories, Richmond, CA).
Band densities were quantified with a GS 800 Densitometer (Bio-Rad Laboratories, Richmond, CA) using Molecular Analyst® image analysis software (Bio-Rad Laboratories, Richmond, CA).
7
Quantitative Western Blot Analysis of Thylakoid Membrane Proteins
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For Western blot analysis, the proteins in the thylakoid membranes were solubilized and separated by commercial linear-gradient SDS polyacrylamide gels (Shaanxi ZHHC Bio-medical Technology Co., Ltd., Xi’an City, China), including 12% universal PAGE for most subunits and 16% special PAGE for detecting PsbE, PsbH, PsbI, PsbN, PsbR, PsaD, and AtpH because of their lower molecular weights. The proteins were transferred onto nitrocellulose membranes (0.2 μm, GE) using the tank transfer systems offered by Mini Trans-Blot (Bio-Rad Laboratories (Shanghai) Co., Ltd., Shanghai, China) and probed with 27 specific primary antibodies (commercial products from Agrisera (Vännäs, Sweden) and kindly supplied by Dr Lixin Zhang). All the antibodies were diluted in 1:2000. Signals from the secondary conjugated antibodies were detected by the enhanced chemiluminescence method using Fujifilm LAS3000 according to the published methods [18 (link)]. The band intensities were quantified with Image J [59 (link)].
8
Quantification of Hepatic Transporter Proteins
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Total protein in the liver was extracted with RIPA Lysis Buffer (Beyotime) containing 1 mM PMSF, and the total protein concentration was determined using the BCA protein assay kit. An equal amount of proteins (50 μg) was separated by electrophoresis on sodium dodecyl sulfate/8.75% polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Millipore, United States) for 2 h through wet transfer method using Mini Trans-Blot (Bio-Rad, United States). Subsequently, the membranes were blocked with 5% nonfat milk dissolving in 0.5% TBST buffer for 1.5 h and then incubated overnight at 4°C with specific primary antibodies including Bsep (goat, Santa Cruz Biotech), Mrp2 (rabbit, Abcam), and β-actin (mouse, Proteintech). After washing the membrane with 0.5% TBST buffer for three times, the membranes were further incubated with corresponding secondary antibodies at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence detection system (Millipore, United States).
9
Quantitative Western Blot Analysis of PAFR and PKA-Cα
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Proteins were subjected to Coomassie blue quantification before use in western blotting with some modification of published methods (Ibe et al. 2000 , 2002 , 2007 ). Proteins were suspended in sodium dodecyl sulfate (SDS) sample buffer, pH 6.8, containing: 125 mmol/L Tris‐base, 4% SDS, 0.006% bromophenol blue, 36 mmol/L EDTA, 90 mmol/L DTT, 10% glycerol, 10% beta‐mercaptoethanol. Proteins were loaded on each sample per lane and subjected to SDS‐PAGE gel electrophoresis for 1 h at 200V on 4–12% Tris‐glycine gradient gels (Lonza, Rockland, ME), along with Bio‐Rad kaleidoscope prestained molecular weight markers and protein standards. After 1 h of SDS‐PAGE, proteins were transferred to nitrocellulose membrane by means of mini Trans‐Blot, (Bio‐Rad, Irvine, CA) at 70V and then blocked with 5% nonfat dry milk in 1% T‐TBS overnight. Blots were then incubated for 2–4 with the specific antibody, for instance, 1:500 of PAFR antibody; then, each antibody was washed with 1% T‐TBS, incubated for 1 h with an anti‐rabbit Ig HRP‐linked secondary antibody (Amersham), followed by three more washes with 1% T‐TBS. The signals were developed for 1 min using Amersham ECL Western Blot detection kit and then exposed to X‐ray film. Bands corresponding to PAFR and PKA‐Cα proteins were scanned and quantified.
10
Western Blot Analysis of His-tagged Proteins
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Proteins were fractionated by 12% SDS-PAGE under reducing conditions and were transferred from the gel to nitrocellulose membranes (Hybond N, GE Healthcare, Chicago, USA) with the aid of a Mini Trans-Blot apparatus (BioRad, Richmond, CA, USA). The immunoblot was essentially performed as described [24 (link)]. The membranes were incubated with mouse monoclonal anti-histidine tag antibodies (anti-His tag, GE Healthcare, Chicago, USA) at a final dilution of 1:1,000. After 1 h at RT, membranes were washed, and goat anti-mouse Immunoglobulin G (IgG) coupled to peroxidase was added to the membranes at a final dilution of 1:2,000 (Sigma-Aldrich, St. Louis, MO, USA). After 1 h incubation at RT, the reaction was developed using a chemiluminescence detection assay (ECL, GE Healthcare, Chicago, USA).
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