Briefly, for RIP assays, cell lysates were prepared in RIP buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.1% Nonidet P-40 5% glycerol 0.5 mM DTT) supplemented with RNase inhibitor. IRF3 antibodies using immunoglobulin G control were incubated with cell lysates overnight at 4 °C. Then, 50 µL Protein A/G Magnetic Beads (MedChemExpress) were added and incubated with the mixture for 2 h. Coprecipitated RNAs were extracted and analyzed by RT–qPCR. The primers used are listed in SI Appendix, Table S1 . For the coIP assay, cell lysates were prepared by using lysis buffer (50 mM Tris × HCl pH 7.0, 10 mM EDTA, 1% SDS, 5 µL/mL protease inhibitor). Then, the cell lysates were mixed with the primary antibody and Protein A/G Magnetic Beads (MedChemExpress) overnight at 4 °C. After that, the beads were washed three times using PBS, followed by Western blot analysis.
Protein a g magnetic beads
Manufactured by MedChemExpress
Sourced in United States, China
Protein A/G Magnetic Beads are a type of lab equipment used for the purification and separation of antibodies from biological samples. These beads are made of a magnetic core coated with a layer of Protein A and Protein G, which have a high affinity for immunoglobulins. The magnetic properties of the beads allow for easy separation and isolation of the desired antibodies using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ip lysis buffer, by Thermo Fisher Scientific (11 mentions)
Np 40 lysis buffer, by Beyotime (4 mentions)
Fast silver stain kit, by Beyotime (3 mentions)
B900610, by Proteintech (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ip lysis buffer, by Beyotime (2 mentions)
Triton, by Bio-Rad (2 mentions)
Mg132, by MedChemExpress (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti flag, by Merck Group (2 mentions)
Ab172730, by Abcam (2 mentions)
Anti ha magnetic beads, by MedChemExpress (2 mentions)
Ip lysis solution, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Anti flag magnetic beads, by MedChemExpress (2 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
82 protocols using protein a g magnetic beads
1
RIP and Co-IP Assays for IRF3 Interactions
2
Magnetic Bead-Based Protein-RNA Isolation
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Protein A/G Magnetic Beads (MedChemExpress, Shanghai, China) were washed three times with TBST. The beads were incubated with the antibody in cell lysis buffer (Beyotime Biotechnology) for 20 min at room temperature on a shaker. Following this, yeast RNA (Beyotime Biotechnology) and FBS (AusgeneX, Oxenford, Australia) were added to the beads, and the beads were incubated for another 20 min. The beads were washed three times with TBST. Subsequently, cell lysates were added to the beads and then incubated on a shaker for 30 min at room temperature. Following incubation, the beads were washed three times with TBST. Then, “RLT Plus Lysis Buffer” from the “SPARKeasy Improved Tissue/Cell RNA Kit” (Sparkjade Biotechnology) was added to the beads. The samples were ready for RNA isolation using the “SPARKeasy Improved Tissue/Cell RNA Kit”.
3
Protein A/G Magnetic Bead Immunoprecipitation
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Protein A/G Magnetic Beads (HY‐K0202, MedChemExpress) were washed four times with 0.5% PBS‐Triton X‐100. The antibody solution was then added, and the magnetic beads and antibodies were incubated for 1 h at 25°C on a flip mixer. The antibody‐conjugated magnetic beads were washed four times with 0.5% PBS‐Triton X‐100. Prepared protein supernatant was then added, and the antibodies and proteins conjugated to the magnetic beads were incubated for 2 h at 4°C on a flip mixer. After washing with 0.5% PBS‐Triton X‐100, 1 × SDS‐PAGE protein loading buffer was added; samples were denatured by heating, and proteins were measured by WB.
4
Proteomic Identification of S100A9 Interactome
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After transfection with the S100A9 overexpression plasmid (S100A9) for 24 h, H6C7 cells were harvested and lysed. Anti-S100A9 or IgG antibodies (Abcam; Cambridge, UK) were added to the lysis solution for antibody immobilization at 4 °C overnight. After incubation with Protein A/G Magnetic Beads (MedChemExpress; Shanghai, China) at 4 °C for 3 h, the protein complex was centrifuged and then washed 3 times with Pierce IP Lysis Buffer (Thermo; MA, USA) for SDS-PAGE analysis. Finally, the SDS-PAGE gel was subjected to silver staining to detect the difference in protein binding between the S100A9 and IgG antibodies. In addition, after pull-down experiments, the two protein samples underwent reductive alkylation and enzymolysis. Moreover, to detect the polypeptide sequence of protein samples, LC-MS/MS analysis was implemented. The polypeptide sequence was identified using ProteinPilot software of the AB SCIEX Triple TOF™ 5600 plus MS system (MA, USA).
5
Immunoprecipitation of Ubiquitin and SF3A3
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Transfected cells were lysed with Pierce™ IP Lysis Buffer (Invitrogen, CA, USA). Protein concentrations were measured with a BCA protein assay kit (Beyotime, China) and the lysates were incubated with primary antibody for Ubiquitin HA-tag (Abcam) or SF3A3 (Abcam) with protein A/G magnetic beads (MedChemExpress, USA) at 4 °C overnight. The beads were washed using IP lysis buffer for 6 times before immunobloting by the indicated antibodies.
6
Immunoprecipitation and Western Blot
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Cells were lysed in RIPA lysis buffer (CST, USA) containing protease inhibitors (Calbiochem) for 30 min on ice and were then centrifuged at 12,000 rpm for 15 min. The supernatant was incubated with antibody for 1 h at 4 °C and the lysate-antibody complexes were incubated with Protein A/G Magnetic Beads (MedChemExpress) for 6 h at 4 °C. The precipitated agarose was washed four times with lysis buffer to remove nonspecific binding. The immune complex was eluted with 2 × SDS Loading buffer and boiled, separated on SDS-PAGE and analyzed by western blot assay.
7
Chromatin Immunoprecipitation and Western Blotting
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After treatment, the cells were lysed with moderate IP buffer (NP-40, Beyotime) with 1% protease inhibitors (Servicebio) and 1.5% phosphatase inhibitor (MedChemExpress) at 4 °C for 20 min and centrifuged at 14,000 × g for 15 min at 4 °C to separate the soluble fraction. The resultant supernatants were incubated with anti-dsDNA or IgG isotype control monoclonal antibody at 4 °C for 8 h. Then, 1 mg protein was incubated with 5 μg antibodies. Protein A/G magnetic beads (MedChemExpress) were coincubated with the antigen-antibody mixture for 2 h at 4 °C. The beads were washed and divided into two parts. Half of the beads were eluted with SDS loading buffer (Boster) and then boiled at 95 °C for 10 min for western blotting analysis, while the other half were eluted with chromatin immunoprecipitation (ChIP) elution buffer (5 mM Tris-HCl, 10 mM EDTA and 1% SDS). After proteinase K treatment, DNA was collected using a TIANamp Genomic DNA Kit (Tiangen), followed by general PCR analysis as described above18 (link),39 (link).
8
Protein Extraction and Immunoprecipitation Protocol
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Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland), as previously described [16 (link)]. Equal amounts of protein were separated by SDS–PAGE and transferred to membranes. For immunoprecipitation, cell lysates were incubated with antibodies and Protein A/G Magnetic Beads (HY-K0202, MedChem Express), followed by western blotting. Western blotting was performed as previously described [19 (link)] with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies, followed by visualization using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel). The antibodies used were as follows: anti-cyclin G2 (DF2284, Affinity Biosciences), anti-Flag (M20008XS, Abmart), anti-PP2Ac (2038 T, Cell Signaling Technology), anti-STAT1 (14994S, Cell Signaling Technology), anti-p-STAT1 (Y701) (9167S, Cell Signaling Technology), anti-lamin B1 (sc-6216, Santa Cruz), anti-β-tubulin (M30109XS, Abmart), anti-GAPDH (M20006F, Abmart), and anti-IgG (3900S, Cell Signaling Technology).
9
Co-immunoprecipitation of IP3R1 and VDAC1
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Co-immunoprecipitations (Co-IP) was performed as previously with minor adaptations [25 (link)]. The RMECs were lysed for 30 min using Pierce™ IP lysis buffer (Thermo Fisher, Waltham, MA, USA) containing protease inhibitor (Roche, Basel, Switzerland). The lysed cells were centrifuged at 12,000× g for 15 min at 4 °C to obtain the supernatant, and then protein concentration was determined using the BCA, assay kit (Beyotime, Nantong, China). A total of 1/10 of the supernatant was kept as the input group and the remaining 500 μL was taken for immunoprecipitation. Here, the supernatant was incubated with 1 μg primary antibody, IP3R1 or VDAC1, or normal control IgG overnight at 4 °C. The samples were incubated with 50 μL protein A/G magnetic beads (MedChemExpress, South Brunswick, NJ, USA) at 4 °C for 3 h and then centrifuged at 4 °C for 5 min at 1000× g. The magnetic beads were washed five times with buffer, and then proteins were eluted from the beads. The samples were boiled for 5 min in 1 × loading buffer and then resolved in SDS-PAGE. The samples were further analyzed by Western blot.
10
Immunoprecipitation and Western Blot Analysis
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HEK293T cells were transfected with indicated combination of the plasmids. At least 24 h after transfection, the cells were harvested and washed with PBS (Phosphate buffered saline). The cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1.5 mM MgCl2, 0.2% (v/v) NP-40, 5% (v/v) glycerol, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)) and sonicated (4°C, 50 W, 5 sec on, 5 sec off, 2 min). The samples were centrifuged at 12,000 g for 10 min. The protein A/G magnetic beads (MedChemExpress, NJ) were incubated with a Myc antibody (Medical and biological Lab, Japan) for 2 h at 4°C, followed by incubation with the supernatants of the samples overnight at 4°C. The beads were then washed three times in the lysis buffer. An aliquot (50 μl) of lysis buffer and the same amount of 2x Laemmli sample buffer were added to the beads. After boiling for 10 minutes at 100°C, a fraction of the input lysate and the immunoprecipitation samples were analysed by western blot analysis.
The protein samples were separated by SDS-PAGE, followed by immunoblot analyses with the indicated antibodies and detection with the ECL (Enhanced chemiluminescent) substrate (Thermo Fisher Scientific, MA).
The protein samples were separated by SDS-PAGE, followed by immunoblot analyses with the indicated antibodies and detection with the ECL (Enhanced chemiluminescent) substrate (Thermo Fisher Scientific, MA).
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