Western blotting was performed as previously described23 (link). In brief, 10 mg of tissue was added to 500 µl ice cold radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor (Roche) and homogenized on ice using a rotor-stator homogenizer (IKA) followed by centrifugation at 13,000 rpm at 4 °C for 30 min. Protein lysates were incubated in 4x Roti load sample buffer (Roth) for 5 min at 90 °C and resolved by SDS-PAGE (12% gels). Proteins were then transferred to PVDF membranes (Bio-Rad) using the Mini-Protean system (Bio-Rad). After blocking nonspecific binding, PVDF membranes were incubated with primary antibodies, including chicken anti-GFP (Aves Labs; 1:2000) for eGFP-L10a detection. Anti-GAPDH (Bethyl Labs, 1:5000) and anti-β-actin antibody (Santa Cruz, 1:10000) served as loading control. Membranes were subsequently probed with HRP-conjugated secondary antibody (Santa Cruz Biotechnology/ Bethyl Labs, 1:10000). Chemiluminescent detection of Proteins was performed using ClarityTM ECL substrate (Bio-Rad) on the Fusion FX (Vilber) chemiluminescent imaging platform.
Roti load sample buffer
Manufactured by Carl Roth
Sourced in Germany
Roti-Load sample buffer is a premixed solution designed for preparing protein samples prior to gel electrophoresis. It contains the necessary components to denature and solubilize proteins, as well as a tracking dye to monitor sample migration during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean 2, by Bio-Rad (2 mentions)
Image lab program, by Bio-Rad (2 mentions)
Ultrasonic probe, by Bioventus (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Lumi lightplus western blotting substrate, by Roche (2 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Fortis Life Sciences (1 mentions)
Clarity ecl substrate, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Chicken anti gfp, by Aves Labs (1 mentions)
Fusion fx, by Vilber (1 mentions)
Roti nanoquant, by Carl Roth (1 mentions)
Polyvinylidene fluoride (pvdf), by Carl Roth (1 mentions)
Ab92446, by Abcam (1 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
10 protocols using roti load sample buffer
1
Quantitative Western Blotting Analysis
2
Western Blot Analysis of Cockroach Brain Proteins
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Cockroach brains were homogenized in 150 μL Roti-load sample buffer (Roth, Karlsruhe, Germany) and incubated for 5 min at 95 °C. Alternatively, membrane proteins were isolated as described previously [61 (link)] and incubated for 5 min at 60 °C. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 10 μg protein, as determined by a modified Bradford assay (Roti-Nanoquant, Roth, Karlsruhe, Germany), was run per lane and then transferred to polyvinylidene fluoride membranes (PVDF, Roth, Karlsruhe, Germany). PVDF membranes were labeled with antibodies as described previously [61 (link)] by applying anti-PeaDOP2 (1:8000) followed by incubation with a secondary antibody conjugated to horseradish peroxidase (1:20,000; American Qualex, La Mirada, CA, USA). For controls, antibodies were pre-absorbed with the fusion protein (15 μg/mL) used for immunization. Immunoreactivity was detected by enhanced chemifluorescence.
3
Immunoprecipitation of TRIM5 and CYPA
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The GST-CYPA-based pulldown experiments followed a protocol previously described (43 (link)). To confirm the expression of primate TRIM5 proteins, cells were lysed using RIPA lysis buffer [25 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% NP-40, 1% glycerol, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitor cocktail set III (Calbiochem, Darmstadt, Germany). The cell lysate was centrifuged at 14,800 rpm for 20 min at 4 °C. The protein supernatant was denatured using Roti-load sample buffer (Roth, Karlsruhe, Germany) and was used for SDS polyacrylamide gel electrophoresis (PAGE). The following primary antibodies were used: anti-HA (mouse MMS-101P, Covance, Münster, Germany, 1:7,500 dilution) for HA-tagged proteins, anti-human TRIM5α (rabbit monoclonal, # 143265; Cell Signaling Technology Europe BV, Frankfurt, Germany, 1:1,000 dilution), anti-CYPA (mouse monoclonal; Santa Cruz Biotechnology, 1:500 dilution), anti-GST (mouse SAB4200237-200 UL, Sigma-Aldrich, Germany,1:7,500 dilution). For viral proteins, viral supernatant was centrifuged through 20% sucrose gradient for a minimum of 4 h at 4 °C, 14,800 rpm. The supernatant was discarded, and the viral pellet was lysed using RIPA buffer for 5 min followed by denaturation at 95 °C in Roti-load sample buffer for 5 min and SDS PAGE using anti-p24 antibody (NIH).
4
SATB2 Immunoprecipitation from Cortical Tissue
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Neonatal cortical tissue was homogenized using a dounce homogenizer in IP lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP‐40, 1 mM EDTA, 5% glycerol; Pierce). DIV13 cortical cultures were lysed on the culture dish in IP lysis buffer (Pierce). The lysates were incubated for 10 min on ice while shaking, followed by a brief centrifugation at 13,000 g. The supernatant was used in immunoprecipitation reactions using the Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 50 µl of protein G Dynabeads was coated with 5 µg of anti‐SATB2 antibody (ab92446, Abcam), and beads were mixed with 500 µg of cortical lysate and incubated overnight at 4°C. On the next day, the beads–antibody–protein complexes were washed three times with washing buffer, re‐suspended in 2 × Roti‐Load sample buffer (Roth) for elution, and incubated at 95°C for 5 min. The eluates were separated by SDS–PAGE and used for further immunoblotting analysis.
5
Western Blotting Protein Detection
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Total protein lysates were prepared in 2 × Roti‐Load sample buffer (Roth). Western blotting was performed as described previously (Loy et al, 2011 ). Membranes were blocked with 5% milk in TBST (0.1% Tween 20 in TBS) for 1 h and then incubated overnight at 4°C with the corresponding primary antibodies diluted in blocking solution. After incubation with HRP‐coupled secondary antibodies, the blots were incubated with ECL reagent (Bio‐Rad) and developed using the BioRad chemiluminescence detection system (ChemiDoc™).
6
Quantitative Western Blotting of Neuronal Proteins
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Extracts of total protein were prepared by lysis of neurons on the cell culture dish in 2 x Roti-Load sample buffer (Carl Roth, Germany). Western blotting was performed as described previously (Loy et al., 2011 (link)). Membranes were blocked with 5% milk powder in TBST (0.1% Tween 20 in TBS) for 1 hr and then incubated overnight at 4°C with the corresponding primary antibodies, diluted in blocking solution. After incubation with HRP-coupled secondary antibodies, the blots were developed using ECL reagent (GE Healthcare, Chicago, IL). Blots were either developed using CP100 photo-developer (Agfa, Belgium) or FUSION-FX7 chemiluminescence detection system (Vilber Lourmat, France). Quantification of protein expression was carried out either by using ImageJ (RRID:SCR_003070 ) or FUSION-CAPT image analysis software in the linear range of detection.
7
Western Blotting Technique for Protein Detection
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Western blotting procedures were described previously83 (link). Briefly, cells were lysed in 250 µL Roti-Load sample buffer (ROTH, Karlsruhe, Germany), preheated to 95 °C and then boiled for an additional 10 min before loading on SDS gel electrophoresis. Primary antibodies were diluted 1:4000 for anti-sGCα1 and 1:2000 for anti-sGCβ1 antibody in 3% dry milk in TBST and incubated with nitrocellulose membranes at 4 °C over-night following challenge of membranes with secondary goat anti-rabbit antibody (1:2000 in 3% milk in TBST) conjugated to horseradish peroxidase (Dako A/S, Denmark). Immuno-complexes were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Freiburg). Samples were quantified with a Kodak Imager Station 440 CF and with the NIH 1.6 software, and all blots were standardized to ß-actin or GAPDH expression that was not affected by the treatments. Representative western blot examples are shown in Supplementary Fig. S4 .
8
Orai1 Expression Analysis in TAC-BSA Treated Cells
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Cells were incubated with with TAC-BSA (100 nM, Sigma) for the indicated time periods, washed twice with ice-cold PBS and suspended in ice-cold lysis buffer (50 mM Tris/HCl, 1 % TritonX-100 pH 7.4, 1 % sodium deoxycholate, 0.1 % SDS, 0.15 % NaCl, 1 mM EDTA, 1 mM sodium orthovanadate) containing a protease inhibitor cocktail (Sigma). The protein concentration was determined using the Bradford assay (BioRad). 40 μg of total proteins were boiled with Roti-Load sample buffer (Carl Roth, Germany) for 5 min at 95 °C and separated in 10 % SDS-PAGE. Proteins were transferred to a PVDF-membrane (Thermo Fisher Scientific, USA) and blocked for 1 h at room temperature with 5 % BSA (Carl Roth, Germany) in TBST. For immunostaining membranes were incubated overnight at 4 °C with anti-Orai1 (1:1000, Cell Signaling) and GAPDH (1:3000, Cell Signaling, USA) antibodies. To detect the specific proteins membranes were incubated for 1h at RT with a 1:2000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, USA). After washing, bands were visualized using the ECL western blotting detection reagent (GE Healthcare, USA) and quantified by Quantity One Software (ChemiDoc XRS, Bio-Rad, USA).
9
Protein Isolation from Mammalian Cell Cultures
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To isolate proteins from monolayer cell cultures, medium was aspirated, cells were washed with PBS, and subsequently lysed in 1x Roti-Load sample buffer (Carl Roth, Karlsruhe, Germany) with additional homogenization using an ultrasonic probe (Misonix, Farmingdale, NY, USA). Lysates were incubated at 90 °C for 5 min and cleared by centrifugation (1 min, 10,000 g). 15 μl of the protein lysates were separated using SDS-8%-PAGE and blotted onto nitrocellulose membranes (Schleicher & Schüll, Dassel, Germany) in a tank blot unit (Mini-PROTEAN II, BioRad, Hercules, CA, USA). After blocking with a 3% BSA solution, membranes were incubated with primary antibodies, washed, and incubated with HRP-conjugated secondary antibodies. After adding Lumi-Light plus Western Blotting Substrate (Roche Diagnostics, Mannheim, Germany), chemiluminescence was recorded using a ChemiDoc MP system and evaluated using the Image Lab program (both from Bio-Rad).
10
Protein Isolation and Western Blotting
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To isolate proteins from monolayer cell cultures, medium was aspirated and cells washed with phosphate-buffered saline (PBS), and subsequently lysed in 1x Roti-Load sample buffer (Carl Roth, Karlsruhe, Germany) with additional homogenization using an ultrasonic probe (Misonix, Farmingdale, NY, USA). Lysates were incubated at 90°C for 5 minutes and cleared by centrifugation (1 minute, 10.000 g). Protein lysates were separated using SDS-8%-PAGE and blotted onto nitrocellulose membranes (Schleicher & Schüll, Dassel, Germany) in a tank blot unit (Mini-PROTEAN II, BioRad, Hercules, CA, USA). After blocking with a 3% BSA solution, membranes were incubated with primary antibodies reacting with: Robo1 (1:1000; Cat.No. ab7279), fascin (1:3000: Cat.No. ab78487) (abcam, Cambridge, UK), vimentin (1:1000; Cat.No. 9782), focal adhesion kinase (FAK; 1:1000; Cat.No. 3285), Phospho-FAK (Tyr925; 1.1000; Cat.No. 3284) (Cell Signaling Technology, Frankfurt, Germany), and β-actin-POD (1:25000; Cat.No. A3854, Sigma-Aldrich, St. Louis, USA). HRP-conjugated secondary antibodies (Cell Signaling Technology) and the LumiLight plus Western Blotting Substrate (Roche Diagnostics, Mannheim, Germany) were used. Chemiluminescence was recorded using the ChemiDoc MP system and Image Lab program (Bio-Rad, München, Germany).
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