Skeletal muscle fibers were isolated from adult Wistar rats. Animals were anesthetized and sacrificed by procedures that are in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003) and approved by the Institutional Animal Care and Use Committee (IACUC) of College of Medicine, National Taiwan University. In brief, vastus lateralis (thigh muscle) was dissected and homogenized in the T-PER tissue extraction reagent (Thermo Scientific; 0.1 g muscle per 500 μl reagent). Lysates were cleared by micro-centrifugation at 13,000 rpm for 15 min. The supernatants were pre-cleared with protein A sepharose beads (GE Healthcare Biosciences) for 2 hrs, incubated with the anti-CLC-1 antibody at 4 °C for 2 hrs, and then incubated with protein A sepharose beads at 4 °C for 16 hrs. Beads were gently spun down and washed twice in the T-PER reagent and then three times with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer. Where indicated, the specificity of immunoprecipitation was verified with mouse IgG (Sigma).
Protein a sepharose beads
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden, New Zealand, Germany
Protein A-Sepharose beads are a widely used chromatographic resin primarily used for the purification and isolation of immunoglobulins and other proteins that bind to Protein A. The beads consist of Sepharose, a cross-linked agarose-based matrix, with Protein A covalently immobilized on the surface.
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Lab products found in correlation
Complete mini, by Roche (18 mentions)
Protein assay reagent, by Bio-Rad (13 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
Du 800 spectrophotometer, by Bio-Rad (9 mentions)
Complete protease inhibitor cocktail, by Roche (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Image studio software, by LI COR (6 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Du800 spectrophotometer, by Beckman Coulter (5 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (5 mentions)
Odyssey scanner, by LI COR (5 mentions)
Protease inhibitor, by Roche (5 mentions)
Protein assay reagent, by Beckman Coulter (4 mentions)
γ 32p atp, by PerkinElmer (4 mentions)
Cl 69, by Synaptic Systems (4 mentions)
Polyacrylamide gel, by Bio-Rad (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein a sepharose beads, by Santa Cruz Biotechnology (3 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (3 mentions)
319 protocols using protein a sepharose beads
1
Isolation and Immunoprecipitation of Rat Skeletal Muscle CLC-1
2
Kinase Activity Assay for LYN
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Once recovered from Matrigel, 3D cultured cells were lysed in RIPA buffer (50mM Tris/HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 1% Na deoxycolate, 0.1% SDS) supplemented with 1mM Na orthovanadate and protease inhibitor-cocktail (Roche, Burgess Hill, West Sussex, UK). After centrifugation (14000 g for 10 min at 4°C), supernatants (150 μg of protein per sample) were pre-cleared with protein A-Sepharose beads (GE Healthcare, Cardiff, UK) for 45 min at 4°C prior to incubation with anti-LYN antibodies (rabbit polyclonal sc-15) for 2 hr at 4°C. Immunocomplexes were pulled down after binding to protein A-Sepharose beads (GE Healthcare) for 45 min at 4°C and washed twice with 20 mM HEPES, pH 7.4, 5 mM MgCl2, 3 mM MnCl2 1mM, 1mM Na orthovanadate (kinase buffer). Beads were then resuspended in 50 μL of kinase buffer with 2.75 μg of acid denatured enolase (Sigma), 5-10 μCi of γ32P ATP (PerkinElmer, Seer Green, Buckinghamshire, UK) and 1 μM cold ATP. After a 10 min-incubation at 30°C, the reaction was stopped by adding 13 μL of 10mM ATP, 50 mM EDTA and samples were subjected to SDS-PAGE on a 10% acrylamide gel. Gels were fixed in 10% methanol/ 10% acetic acid solution, then dried and developed by autoradiography. Intensities of bands corresponding to phosphorylated enolase were measured using the ImageJ software.
3
Immunoprecipitation and Western Blotting Protocol
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Cells were washed with ice-cold PBS and lysed with IP lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA and 0.5% (v/v) Triton X-100, supplemented with ‘cOmplete’ protease inhibitors (Roche)). The lysates were centrifuged at 50,000 rpm for 30 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with agarose beads (Chromotek), then incubated with GFP-Trap agarose beads (Chromotek) for 1 hr at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C. Samples were subjected to western blotting.
For pulse chase samples, cells were washed with ice-cold PBS and lysed with IP lysis buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1% (w/v) octyl-glucoside, supplemented with protease inhibitors). The lysates were centrifuged at 20,000 g for 20 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with Protein A Sepharose beads (GE Healthcare), then incubated with Protein A Sepharose beads and rabbit anti-caveolin1 antibody overnight at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C.
For pulse chase samples, cells were washed with ice-cold PBS and lysed with IP lysis buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1% (w/v) octyl-glucoside, supplemented with protease inhibitors). The lysates were centrifuged at 20,000 g for 20 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with Protein A Sepharose beads (GE Healthcare), then incubated with Protein A Sepharose beads and rabbit anti-caveolin1 antibody overnight at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C.
4
Immunoprecipitation and Western Blot Analysis of HA-tagged Proteins
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HEK293 cells were transfected with the plasmid vectors and incubated for 48 h. After incubation, the cells were harvested and lysed in buffer A containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40. The cell lysates were clarified by centrifugation at 10,000 rpm for 5 min. After adding 5 µL monoclonal mouse anti-HA antibody, the lysates (300 µL) were incubated at 4 °C for 2 h. Antibody–antigen complexes were mixed with protein A Sepharose beads (GE Healthcare, Uppsala, Sweden) and rotated at 4 °C for a further 8 h. protein A Sepharose beads were recovered by centrifugation and washed 3 times with buffer A. Beads were then suspended in an SDS-sample buffer, and proteins were separated by electrophoresis through a 6% or 10% polyacrylamide gel in the presence of 0.1% SDS. The proteins were transferred to a PVDF membrane and immunoblotted.
5
Chromatin Immunoprecipitation Protocol
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The cultured cells were collected and lysed with RIPA lysis buffer. The antibody against CD31 (ab24590; Abcam) or normal IgG (Sigma-Aldrich) as the immunoprecipitation control was applied to Protein A Sepharose beads (GE healthcare) in PBS and incubated at room temperature for 2 h. Following incubation with lysates at 4°C overnight, the Protein A Sepharose beads were collected and protein–DNA complexes were eluted. The bound DNA was purified with QIAquick columns (Qiagen, Valencia, United States) and subjected to qRT-PCR assay. Primers are listed in Table 1 .
6
Cytosolic and Nuclear Protein Extraction
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Cytosolic fractions and nuclear extracts were prepared using a NE‐PER kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. For immunoprecipitation (IP), cells were lysed in a lysis buffer [20‐mM Tris (pH 7.5), 150‐mM NaCl, 1‐mM EDTA, 1% Triton X‐100, 2.5‐mM sodium pyrophosphate, 1‐mM β‐glycerophosphate, 1‐mM Na3VO4, 1‐mM NaF, and a protease inhibitor mixture]. Cell lysates were precleared using protein A Sepharose beads (GE Healthcare, Piscataway, NJ, USA) and then incubated with anti‐M‐cadherin antibody at 4°C for 1 h. Next, protein A Sepharose beads were added to the lysates and incubated at 4°C for 1 h. Western blotting was performed using the primary antibodies listed in Table S1. For further details, refer to the Supporting information .
7
HLA Class I Peptide Extraction
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1 mL Protein A-Sepharose beads (GE Healthcare) were washed in 50 mM borate, 50 mM KCl (pH 8.0) solution and then incubated with 2 mg of pan-HLA-I antibody (W6/32) slowly rotating for 1 h at 4°C. The beads were washed in a column format with 0.2 M triethanolamine (pH 8.2), and the bound antibody was cross-linked by incubation with 40 mM dimethyl pimelimidate dihydrochloride (DMP) (Sigma) (pH 8.3) for 1 h at room temperature. Ice-cold 0.2 M Tris buffer (pH 8.0) was added to the mixture to stop the reaction. Unbound antibody was washed off the column by washing with 0.1 M citrate (pH 3.0), and the column was equilibrated in 50 mM Tris (pH 8.0) for further use. 5 × 108 cells pellets were lysed by using 10 mL lysis buffer (0.5% IGEPAL 630, 150 mM NaCl, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail (Roche)), and mixed for 30 min. The lysate was centrifuged at 300 g for 10 min to remove nuclei and then at 15,000 g for 60 min to pellet other insoluble material. 1 mL W6/32 cross-linked to Protein A-Sepharose beads (GE) was added to cleared lysates for 1h, and beads were washed with 50 mM Tris buffer (pH 8.0) containing first 150 mM NaCl, then 450 mM NaCl, and next no salt. HLA-peptide complexes were eluted by using 5 mL 10% acetic acid and dried.
8
Pha13 Immunoprecipitation and Ubiquitination
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The IP assay for Pha13 was performed using total proteins extracted from 0.25 g of Phalaenopsis orchid leaves with 200 ul immunoprecipitation buffer (50 mM Tris-pH 7.5, 0.1% NP40, 10 mM MgCl2, 150 mM NaCl, 10 uM MG132, 1X complete protease inhibitor [Roche]). The extract was centrifuged at 12000 ×g for 10 min at 4 °C. Then, the supernatants were transferred to the non-stick tube, and pre-cleared with protein A sepharose beads (GE Healthcare Life Sciences) 1 hours at 4 °C with gentle shaking. After, the extract was centrifuged at 1500 ×g for 2 min at 4 °C, the supernatant was further incubated with the anti-Pha13 antibodies for 2 hours at 4 °C with gentle shaking, followed by incubation with protein A sepharose beads (GE Healthcare Life Sciences) for 2 hours at 4 °C with gentle shaking. The beads were washed three times with ice-cold immunoprecipitation buffer and eluted by SDS-PAGE sampling buffer. The eluted proteins were analyzed by immunoblotting using the anti-Pha13 antibody and anti-ubiquitin antibody (Research and Diagnostic Systems).
9
Kinase Activity Assay for LYN
10
Investigating MBP-TcpF Interaction with MyD88
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Recombinant MBP, MBP-TcpF or MBP-TcpF mutant (MBP-TcpFm) fusion protein purified from E. coli, was mixed with 1 mg RAW264.7 cell lysate overnight at 4°C under agitation. The mixture was precleared with protein A Sepharose beads (GE Healthcare, Piscataway, NJ) by rotation for 1 hour followed by incubation with anti-MBP antibody and protein A Sepharose beads for 4 hours. The immune complexes were collected by spinning at 500× g for 5 min. The beads were washed three times in lysate buffer before being subjected to polyacrylamide gel electrophoresis, followed by transfer to PVDF membrane for immunoblotting with MyD88 specific antibody (Cell Signaling Technology, Beverly, MA).
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