Venom samples (200 µg) submitted to BN/SDS-PAGE were transferred to nitrocellulose membranes in a semi-dry system (Bio-Rad, USA) at 15 V for 2 h. Membranes were blocked with 5% non-fat milk in washing buffer (0.1% Tween 20 in PBS, pH 7.4), incubated at room temperature with polyclonal anti-botrocetin antibodies (1:20,000) for 2 h, and subsequently incubated with AlexaFluor 647-conjugated anti-rabbit IgG (1:2500) for 1 h. The membranes were analyzed in a ChemiDoc MP system (Bio-Rad, USA) and the ImageLab software (version 5.2.1, Bio-Rad, USA) was used to observe reactive spots [61 (link),69 (link)]. Thereafter, the same membrane was blocked again with 5% non-fat milk and incubated with Bothrops antivenin (1:1000), and subsequently with peroxidase-conjugated anti-horse IgG (1:8000). The reaction was developed with diaminobenzidine (DAB) as previously described [70 (link),71 (link)].
Semi dry system
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Switzerland
The Semi-dry system is a lab equipment designed for the transfer of proteins from a gel to a membrane during the Western blotting process. It utilizes a simple and efficient electrophoretic transfer method to facilitate the movement of proteins from the gel to the blotting membrane.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Rabbit anti parp, by Cell Signaling Technology (2 mentions)
Protran ba 83, by GE Healthcare (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (2 mentions)
Chemi lumi one super, by Nacalai Tesque (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Rabbit anti vinculin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Skim milk, by BD (1 mentions)
46 protocols using semi dry system
1
Western Blot Analysis of Snake Venom Proteins
2
Analyzing Phosphorylated p38 Protein Levels
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Cell suspensions and lung lysates in the electrophoresis sample buffer were heated at 100°C for 5 min and run in 10% polyacrylamide gels (PAGE-SDS). After transfer of the proteins to nitrocellulose membranes at 15 V for 60 min (Biorad semidry system), the membranes were incubated with a blocking solution followed by incubation with a monoclonal antibody against phosphorylated p38 (Cell Signaling; 1:1000 dilution) and then with a peroxidase-conjugated anti-mouse antibody (Pierce; 1:10,000). Detection was performed by utilizing the “Super Signal Chemiluminescence” kit (Pierce) and then exposing the membrane to an autoradiograph film (Kodak MR Biomax). Membranes containing proteins were stripped, blocked again, and incubated with monoclonal antibodies to total p38 (Cell Signaling; 1:1000) or glycerol-3-phosphate dehydrogenase (GAPDH) followed by treatment with an anti-mouse antibody conjugated to peroxidase. After digitalized and analysis by size and intensity using the Image Master 2D Elite 4.01 equipment, the bands were compared to those of the controls and normalized against total p38 or GAPDH. The expression results are in folds over the controls.
3
Protein Extraction and Western Blot Analysis
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The cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 1 mM EGTA, 1 mM EDTA, 0.25% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Roche Diagnostic, Mannheim, Germany). Proteins were resolved by 10% SDS PAGE (about 30 μg of extract per lane was loaded) and transferred onto a nitrocellulose membrane (Protran BA83, GE Healthcare, Chicago, IL, USA) using a semi-dry system (Bio-Rad Laboratories S.r.l., Segrate, Italy). Blocking and antibody incubations were performed at room temperature in TBS containing 0.1% Tween 20 and 5% low fat milk for 1 h. The following antibodies were used: rabbit anti-PARP (Cell Signaling Technology, Danvers, MA, USA), mouse anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-rabbit-pS15 p53 (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-Vinculin (Cell Signaling Technology, Danver, MA, USA). HRP-conjugated secondary antibodies (Bio-Rad Laboratories S.r.l., Segrate, MI, Italy) were revealed using the Clarity Western ECL Substrate (Bio-Rad Laboratories S.r.l., Segrate, Italy).
4
Western Blot Protein Detection Protocol
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The recombinant proteins were separated on 12% SDS-PAGE and subsequently transferred to a 0.22-μM-pore nitrocellulose filter membrane (NC, Merck Millipore, Tullagreen, Carrigtwohill, Ireland) using a semi-dry system (Bio-Rad, Hercules, CA, USA). The transferred membrane was blocked with 5% skim milk (BD, Baltimore, MD, USA) diluted in TBS (Tris buffered saline) with 0.5% Tween-20 (TBST) for 2 h at 37 ℃ and incubated with goat serum diluted by 1:100 in blocking buffer for 1 h at 37 °C. After 3 washes with TBST, the strips were probed with a 1:4000 diluted secondary antibody, horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Thermo Fischer Scientific, Waltham, MA, USA). Then, the strips were washed with TBST five times and the immunoreactions were evaluated using High-sig Electro-Chemi-Luminescence (ECL, Thermo Fischer Scientific, Waltham, MA, USA) Western blotting substrate.
5
MBNL1 Protein Quantification by Western Blot
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Protein extracts were obtained from 1 × 106 cells after sonication in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Roche Applied Science). Total protein was determined by using Total protein concentration BCA protein assay kit.
(Pierce). For MBNL1 immunodetection 20 mg of total protein was heated 5 min at 100 ºC, electrophoresed on 12% SDS-PAGE gels, and transferred using a semi-dry system (Bio-Rad) onto nitrocellulose membranes (GE Healthcare). Then, membranes were blocked for 1 h at RT in PBS-T (0.05% Tween 20 [pH 7.4]) supplemented with 5% non-fat dried milk and incubated ON at 4 ºC with antibody mouse anti-MBNL1 (1:200, cloneMB1a, The Wolfson Centre for Inherited Neuromuscular Disease, UK) on blocking solution. After three washes with PBS-T membranes were incubated 1 h RT with secondary antibody in blocking solution (anti-mouse-IgG goat horseradish peroxidase (HRP)-conjugated 1:5000, Sigma-Aldrich). β-ACTIN was detected with a primary mouse anti-β-Actin antibody (1 h, 1:5000, Sigma-Aldrich) followed by HRP-conjugated anti-mouse-IgG antibody (1 h, 1:5000, Sigma-Aldrich). Bands were detected using ECL western blotting substrate (Pierce). Images were acquired using ImageQuant LAS 4000 (GE Healthcare).
(Pierce). For MBNL1 immunodetection 20 mg of total protein was heated 5 min at 100 ºC, electrophoresed on 12% SDS-PAGE gels, and transferred using a semi-dry system (Bio-Rad) onto nitrocellulose membranes (GE Healthcare). Then, membranes were blocked for 1 h at RT in PBS-T (0.05% Tween 20 [pH 7.4]) supplemented with 5% non-fat dried milk and incubated ON at 4 ºC with antibody mouse anti-MBNL1 (1:200, cloneMB1a, The Wolfson Centre for Inherited Neuromuscular Disease, UK) on blocking solution. After three washes with PBS-T membranes were incubated 1 h RT with secondary antibody in blocking solution (anti-mouse-IgG goat horseradish peroxidase (HRP)-conjugated 1:5000, Sigma-Aldrich). β-ACTIN was detected with a primary mouse anti-β-Actin antibody (1 h, 1:5000, Sigma-Aldrich) followed by HRP-conjugated anti-mouse-IgG antibody (1 h, 1:5000, Sigma-Aldrich). Bands were detected using ECL western blotting substrate (Pierce). Images were acquired using ImageQuant LAS 4000 (GE Healthcare).
6
Western Blot Analysis of ACADM Protein
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Western blot analysis was performed as previously described (4 (link)). Briefly, RVs were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer containing cOmplete™ protease inhibitor (Roche, Indianapolis, IN), and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Proteins were separated by 12% Tris SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad) using a semi-dry system (Bio-Rad). Membranes were blocked with 5% w/v non-fat dry milk, incubated with primary antibodies (4°C, overnight), and incubated with secondary antibodies at room temperature for 1 h. Proteins were detected by horseradish peroxidase (HRP) chemiluminescence, and lanes were quantified using ImageJ analysis software. An antibody against ACADM (1:10,000; ERP3708) was purchased from Abcam. The vinculin antibody (1:1,000; E1E9V) was purchased from Cell Signaling and was used as a loading control.
7
Immunoprecipitation and Western Blot Analysis
8
Northern Blot Analysis of Small RNAs
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Total RNA was isolated as described above. Northern blot analyses of low molecular weight RNAs were performed by separating 20 μg of total RNA on 14% acrylamide gels. RNAs were transferred to N+ membranes (Amersham Bioscience) by using a semi-dry system (BioRad). For the detection of small RNAs, membranes were hybridized overnight with oligonucleotides complementary to the small RNA sequences. The probes were end labeled with γ-32P-ATP using T4 PNK (New England Biolabs, Beverly, MA) in PerfectHyb Plus hybridization buffer (Sigma). Different temperatures were used for the hybridization according to the different Tm of the probes. The membranes were washed twice for 20 min with 2× SSC, 1% SDS at 40°C, and then scanned on a Typhoon Imaging System 9200 (GE Healthcare) or exposed to x-ray films.
9
Immunoblotting of Recombinant SMYB1
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SDS-PAGE of purified rSMYB1 was performed using 12% gels, and the gels were electroblotted onto nitrocellulose membranes for 30 min at 20 V using a semi-dry system (Bio-Rad). The membranes were blocked with phosphate-buffered saline (PBS) (130 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4) plus 0.05% Tween 20 (PBS-T) containing 5% dry milk (p/v) for 16 h at room temperature. The membrane was subsequently incubated in 1:2000 dilutions of an anti-His antibody (GE Healthcare) and peroxidase-conjugated anti-mouse IgG (Sigma Aldrich) in PBS-T for 1 h at room temperature. After washes using PBS-T, the membrane was developed using 3,3′-diaminobenzidine (Sigma Aldrich), according to the manufacturer's protocol. After developing, the membrane was washed using distilled water and dried on filter paper.
10
Cell Lysis and Protein Extraction Protocol
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HEK293, HeLa, or M17 cells were lysed in radioimmunoprecipitation assay buffer [150 mM sodium chloride, 50 mM tris (pH 8.0), 1% NP-40, 0.5% deoxycholate, 0.1% SDS] supplemented with protease inhibitor cocktail, 1 mM PMSF, and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich). Cell lysates were cleared by centrifugation at 4°C for 15 min at 13,000 rpm. The pellet (insoluble fraction) was resuspended in SDS/TBS (tris-buffered saline) supplemented with protease inhibitor cocktail, 1 mM PMSF, and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich) and sonicated using a fine probe (0.5-s pulse at amplitude of 20%, 15 iterations). BCA protein assay was performed to quantify the protein concentration in the soluble and insoluble fractions before the addition of 4× Laemmli buffer. Proteins from the soluble and the insoluble fractions were then separated on a 16.5% or a 18% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane (Thermo Fisher Scientific) with a semidry system (Bio-Rad), and immunostained.
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