Immunoblotting assay were performed using correspond antibodies for detection of Cx43 and EPAC. Total cell lysates were collected by using ice-cold PBS and homogenization in cold lysis buffer (RIPA buffer). lysate clarified by centrifugation at 12,000 rpm for 10 min, supernatant transferred to a new tube. Protein content was measured using the BCA protein kit (Pierce). Equal amount of protein (25µg) in each sample was loaded to 12.5% standard SDS-PAGE. By using a wet system (Bio-Rad) the proteins were transferred to PVDF membranes. After blocking, membranes were incubated over night with primary antibodies (1/1000), washed three times with TBST 20 (0.5%) incubated with secondary antibody (1/5000) for 1 hour at room temperature. After three washes, detection was performed by applying Pierce ECL Plus Western Blotting Substrate (#32134), emanating chemiluminescence from the membrane for manual x-ray film development. Protein levels were normalized to β-actin.
Wet system
Manufactured by Bio-Rad
Sourced in United States
The Wet system is a laboratory equipment used for the separation and analysis of biological molecules, such as proteins and nucleic acids, through electrophoresis. It facilitates the transfer and immobilization of these molecules onto a solid support, enabling further processing and detection.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Spectra multicolor broad range protein ladder, by Thermo Fisher Scientific (1 mentions)
Nbt bcip, by Roche (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Srx 101a developer, by Konica Minolta (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti clock, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Enhanced hrp dab chromogenic substrate kit, by Tiangen Biotech (1 mentions)
Laminin, by Abcam (1 mentions)
Secondary antibodies conjugated with horseradish peroxidase, by Merck Group (1 mentions)
Fibronectin, by Abcam (1 mentions)
β tubulin, by Abcam (1 mentions)
Type 1 collagen, by Abcam (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
10 protocols using wet system
1
Immunoblotting Analysis of Cx43 and EPAC
2
Western Blot Analysis of Polyhistidine-Tagged Proteins
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For Western blot analysis, the proteins were separated by SDS-PAGE according to the method of Ogita and Markert [42 (link)] and transferred onto a Protran BA 83 nitrocellulose membrane (Whatman, GmBH, Maidstone, UK) by the wet system (Bio-Rad) (200 mA, 90 V for 1 h at 4 °C) in 10 mM CAPS/NaOH buffer, pH 11.0, containing 10% (v/v) ethanol. The SpectraTM multicolor broad range protein ladder (10–260 kDa) (Thermo Scientific, Waltham, Massachusetts, USA) was used as a molecular mass standard. After blocking in TBS (Tris Buffered Saline) buffer containing 5% (w/v) nonfat dry milk, the membrane was incubated with monoclonal anti-polyHistidine antibodies produced in mice (1:12,000) (Sigma-Aldrich). The proteins were detected with goat anti-mouse IgG antibodies conjugated to alkaline phosphatase (1:1000) (Sigma-Aldrich) and visualized using NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) tablets (Roche, Bazylea, Sweden).
3
Western Blot Analysis of Protein Expression
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To perform Western blot experiments, 50 μg of tissue lysate samples were separated using SDS-PAGE and transferred to a 0.45 μm nitrocellulose membrane using the wet system from Bio-Rad (Hercules, California, USA). The membrane was blocked for 1 hour at room temperature using 5% skimmed milk in TBST (see above). For immuno-detection of proteins, membranes were incubated overnight at 4°C with primary antibodies against HABP4 (rabbit, polyclonal, 1:1000 dilution), PCNA (mouse, monoclonal, 1:1000 dilution), Cyclin D1 (rabbit, monoclonal, 1:1000 dilution), or CDK4 (mouse, monoclonal, 1:1000 dilution), diluted in TBST. Three washes of 5 minutes with TBST solution were done and membranes were incubated for 1 hour at room temperature with secondary antibodies anti-rabbit IgG or anti-mouse IgG (Milford, Massachusetts, EUA) (1:5000 dilution), in TBST containing 5% skimmed milk. Three washes of 5 minutes with TBST were done and membranes were developed using the enhanced ECL method using Chemi Doc Imaging System from Bio-Rad. All directly compared samples of e. g. Cyclin D1 from wild-type and HABP4-/- mouse samples (see Figure 5D , second line) were run out side by side on the same gel and blotted to the same blot to guarantee equal processing, exposure times, etc. Only this way it can be guaranteed that the samples can be compared in a semi-quantitative manner.
4
Isolation and Western Blot Analysis of Murine Neutrophils
5
Analysis of CLOCK Knockdown in Human Myotubes
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Human myotubes transfected with siControl or siCLOCK for 24 to 72 hr, were lysed in RIPA buffer. Protein extracts (8 µg) were analyzed by SDS-PAGE and immunoblotted to 0.45 µm nitrocellulose membrane or 0.2 µm PVDF membrane using a wet system (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Membranes were blocked and incubated with primary and secondary antibodies in 5% BSA/TBS-T 0.5% or 5% BSA/TBS-T 0.1%. Primary and secondary antibodies were used at the following dilutions: anti-TBC1D4/AS160 (1/1000, Cell Signaling, Danvers, MA, #2670S), anti-14-3-3θ (1/200, Cell Signaling, #9638S), anti-CLOCK (1/200, Santa Cruz Biotechnology, Santa Cruz, CA , H-276) and anti-ACTIN (1/1000, Sigma-Aldrich, A2066), anti-rabbit-HRP (1:3000, Sigma-Aldrich A8275). Signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). For protein quantification, five donors were analyzed but only the representative western blot result of one donor is shown.
6
Western Blot Analysis of Leishmania TGase
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For the detection of TGase in promastigote lysates, about 14 µg of proteins was separated by 10% polyacrylamide SDS-PAGE under reducing conditions and then transferred onto 0.45 µm pore nitrocellulose membranes using a wet system (Bio-Rad) at 300 mA and 150 V for 90 min using Tris-glycine-SDS blotting buffer with 20% methanol. Non-specific sites of the blots were blocked through incubation with 5% skim milk and 0.5% Tween-20 in PBS (blocking buffer) for 1 hr, and the blots were incubated overnight at 4 °C with rabbit polyclonal antibodies (1:1500 in 1% BSA, 0.5% Tween-20 in PBS) against a conserved region of TGase 2 enclosing a sequence in the catalytic domain of the enzyme. After executing three washing steps with PBS-T (0.1% Tween-20 in PBS), the blots were incubated with anti-rabbit-HRP secondary antibodies (1:50,000 in blocking buffer) for 1 h at RT and washed 3 times for 10 min each with PBS-T and 3 times for 10 min each with PBS. Finally, the bands were detected with the ECL Plex SuperSignal™ West Femto (Thermo Scientific).
7
Immunoblotting of Chlamydomonas Proteins
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The prepared sample of 30 μg proteins were subjected to 12% SDS-PAGE and transferred to PVDF membrane. Following transfer to PVDF, the proteins were visualized by modified colloidal coomassie brilliant blue to verify the protein loading. Membrane blotting was performed with the Wet-system (Bio-Rad, USA) according to the manufacturer's instructions, with a Chlamydomonas reinhartii anti-cytochrome f antibody (1:5000 dilution, Agrisera AB, Swedish) and a plant anti-Lhcb1 antibody (1:5000 dilution, Agrisera AB, Swedish). After incubation with HRP-Goat Anti-Rabbit IgG (H + L) (1:5000 dilution, BOSTER, China), HRP signal was detected with Enhanced HRP-DAB chromogenic substrate kit (TIANGEN, China).
8
Western Blot Analysis of Extracellular Proteins
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Proteins were separated on a 12.5% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane overnight at +4 °C at a constant voltage of 20 V using the Biorad wet system. The Toubin buffer system was used as the transfer buffer. Non-specific binding sites were blocked using 5% BSA in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 1 h. After blocking, the membrane was incubated with primary antibodies specific for ALIX (Abcam, Cambridge, UK; dilution 1/1000), beta-tubulin (Abcam, Cambridge, UK; dilution 1/1000), fibronectin (Abcam, Cambridge, UK; dilution 1/2000), HSP70 (Biocat, Heidelberg, Germany; dilution 1/1000), CD63 (Merck Millipore, Burlington, MA, USA; dilution 1/1000), CD81 (BioLegend, San-Diego, CA, USA; dilution 1/500), collagen type I (Abcam, Cambridge, UK; dilution 1/1000), collagen type IV (Thermo Fischer Scientific, Waltham, MA, USA; dilution 1/1000), or laminin (Abcam, Cambridge, UK; dilution 1/2000) in the blocking solution overnight at +4 °C. The membrane was then washed with TBST and incubated with secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) in the blocking solution. Signal visualization was performed using the ClarityTM Western ECL Substrate kit (BioRad, Hercules, CA, USA) on ChemiDoc (BioRad, Hercules, CA, USA).
9
Western Blot Analysis of Protein Expression
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Cells were lysed with RIPA buffer (Sigma, St. Louis, MO, USA) supplemented with a protease-inhibitor cocktail (Roche, Basel, Switzerland). Samples were incubated for 30 min at 4 °C. After clearing by centrifugation (17,000× g, 15 min), protein concentrations were determined using the DC Protein Assay (Bio Rad, Hercules, CA, USA). Equivalent amounts of protein were separated by electrophoresis in 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose (Amersham Protan, Cytiva, Marlborough, MA, USA) using a wet system (BioRad, Hercules, CA, USA), and analyzed by immunoblotting. The membranes were blocked with 5% BSA and incubated with the specific primary antibodies (the ones used in ICC), and mouse or rabbit HRP-conjugated secondary antibodies. GAPDH (mouse; GTX627408, Genetex, Irvine, CA, USA) and Actin (rabbit; ab8227, Abcam, Cambridge, UK) were used as loading controls. Finally, the membranes were incubated with the Luminata Crescendo Western HRP substrate (Millipore, Burlington, MA, USA), imaged with the ChemiDoc system, and analyzed with associated ImageLab software (Bio-Rad, Hercules, CA, USA). Student’s t-tests were applied for pairwise comparisons.
10
Western Blot Analysis Protocols for Protein Detection
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Protein lysates (30–40 μg) were resolved on 4% to 15% gradient SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane using a wet system (BIO-RAD). Membranes were washed briefly in 1× TBS-Tween (TBST) and blocked in 5% (W/V) dried milk in TBST (1 h), incubated overnight at 4 °C in primary antibodies diluted 1:1000 in 5% (W/V) BSA-containing TBST, washed three times in TBST and incubated in appropriate horseradish peroxidase–conjugated secondary antibody. Membranes were developed using Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and exposed to Blue Lite Autorad Film (GeneMate). GAPDH, actin, or Vinculin was used as loading control. Antibodies used were p53 (7F5, Catalog # 2527S), PUMA (D30C10, Catalog #12450S), p21 Waf1/Cip1 (DCS60, Catalog # 2946S), Gadd45a (Catalog # 4632S), Upf1 (Catalog # 9435), GAPDH (Catalog # 5174) from Cell Signaling Technology, MDM2 (Catalog # sc-56154) from Santa Cruz Biotechnology, and β-actin (Catalog # A5441) and Vinculin (Catalog #V9131) from SIGMA. Protein expressions were normalized to that of GAPDH, β-actin, or Vinculin.
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