The MCF-7 cells ( ) were cultured on dishes and treated with BPA, BPS, or vehicle for 48 h. At the end of treatment, the MCF-7 cells were rinsed three times with ice-cold PBS buffer, followed by incubation for 30 min at 4°C with ice-cold IP lysis buffer (Beyotime) containing the serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Cell lysates were then centrifuged at for 20 min at 4°C. Supernatants were collected and the protein concentrations were determined by a BCA protein assay kit (Beyotime Biotechnology). Cell extracts were incubated with or anti-IgG antibody (at the dilution of 1:1,000) plus protein G–conjugated Sepharose beads (Amersham Pharmacia) in a ratio of of extract per of beads. After overnight rocking at 4°C, the precipitates were collected by centrifugation at for 3 min and washed with ice-cold IP lysis buffer (Beyotime) three times, then subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with , anti-TET2, and anti-SP1 antibodies.
Ip lysis buffer
Manufactured by Beyotime
Sourced in China, United States
IP lysis buffer is a reagent used in the process of cell lysis and protein extraction. It is designed to disrupt cell membranes and release intracellular proteins, making them available for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
M 280 streptavidin magnetic beads, by Merck Group (17 mentions)
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Protein a g agarose, by Beyotime (10 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Protein a g agarose bead, by Beyotime (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (5 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Beyotime (4 mentions)
Protein a g bead, by Santa Cruz Biotechnology (4 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (4 mentions)
Anti flag m2 magnetic bead, by Merck Group (4 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (4 mentions)
Protein g agarose suspension, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Protein a g agarose beads, by Thermo Fisher Scientific (3 mentions)
228 protocols using ip lysis buffer
1
Immunoprecipitation and Western Blotting of ER-α, TET2, and SP1
2
Co-Immunoprecipitation of Alpha-Synuclein and Calpain 1
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A Co-IP assay was conducted as described previously [31 (link)]. After 24 hours of treatment with Mn, brain slices were rinsed three times in ice-cold PBS buffer, followed by incubation for 30 min at 4°C with 1 ml ice-cold IP lysis buffer (Beyotime, Haimen, China) containing the serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Samples were pre-cleared with Protein A+G agarose (Beyotime). Pre-cleared lysates were then incubated with mouse anti-full-length alpha-synuclein monoclonal antibody (1:50) or rabbit anti-calpain 1 polyclonal antibody (1:50) at 4°C overnight. A 25% slurry of Protein A+G agarose was added into the lysates incubated for 2 h at 4°C and washed with ice-cold IP lysis buffer (Beyotime). The pellet was resuspended in SDS loading buffer, boiled for 10 min, and then centrifuged at 12,000 × g for 1 min in a Sigma 3K 30 centrifuge (Sigma, Germany). The supernatant was removed and loaded onto a 12% SDS-PAGE gel separated by electrophoresis and transferred onto a PVDF membrane. Membrane blots were probed with rabbit anti-calpain 1 polyclonal antibody or mouse anti-full-length alpha-synuclein monoclonal antibody and visualized by chemiluminescent detection reagents (Pierce).
3
Immunoprecipitation and Western Blot Analysis
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An optimal quantity of Protein A beads (MCE, America) was placed in a silica tube and subjected to a thrice wash cycle with PBS. The samples were centrifuged at 4 ℃ at 1000 rpm for 2 min. After discarding the supernatant, the beads were washed once with IP Lysis buffer (Beyotime, China). The pH was adjusted to lie between 7. 4–7. 5, and samples were stored at 4℃. Antibodies were added to Protein A beads in IP Lysis buffer, making up the volume to 600 µl, and then incubated under rotation at 4 °C for 3 h. Following centrifugation, the supernatant was removed. The bead-antibody complex was then combined with cell lysate and incubated overnight at 4 ℃. After a series of washes and centrifugation cycles, the appropriate volume of IP Lysis buffer was added, followed by the proportional addition of 1×SDS-PAGE protein buffer (Beyotime, China). The mixture was then boiled for 10 min. For the Western blotting experiment, the primary antibodies used were FEN1 and SUMO2. The resultant binding proteins were subjected to either LC-MS/MS or Western blot analysis, with the primary antibodies being FEN1 and SUMO2 for the latter.
4
Western Blot and Immunoprecipitation Analysis
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Cells were lysed with western and immunoprecipitation (IP) lysis buffers (Beyotime). The lysates were centrifuged at 13 000 × g for 6 min at 4 °C. The supernatants were collected and protein concentrations were determined with a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Equal aliquots of protein samples were loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight, after which they were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Western blots were developed using an enhanced chemiluminescence (ECL) western blotting detection kit (Pierce) and quantified by scanning densitometry. To analyze protein interactions, cell lysates were incubated with Protein A/G agarose beads to perform a preclearing step to reduce nonspecific binding, followed by incubation with primary SOD2 antibody overnight at 4 °C, and the immune complexes were pulled down with fresh Protein A/G agarose beads (Santa Cruz Biotechnology). The bound proteins were eluted in denaturing SDS sample buffer and analyzed by western blotting.
5
Identifying TRIM25 Viral Protein Interactions
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DF-1 cells were seeded in 6-well plates and transfected with the indicated plasmids. Then, the cells were washed thrice with ice-cold PBS and lysed in 500 μL Western blotting and IP lysis buffers (P0013, Beyotime, China) for 30 min. After 12,000×g centrifugation, the supernatants of cell lysates were incubated with 1 μg anti-Flag mouse monoclonal antibody (mAb) or control mouse IgG for 6–8h or overnight. Afterward, 40 μL protein A/G agarose (A10001, Abmart) was added to the lysate mixture for 6–8h. The beads were collected by centrifugation at 3,000 ×g for 5 min at 4 °C and washed five times with ice-cold PBS. To identify the viral proteins related to TRIM25, 2 μg pHA-VP1, pHA-VP2, pHA-VP3, pHA-VP4, or pHA-VP5 and 2 μg pFlag-TRIM25 were co-transfected, respectively, and the search was then conducted following the above instruction. Furthermore, the relationship between TRIM25 and VP3 was detected in both directions by co-transfecting pFlag-plasmids and pMyc-plasmids.
6
Biotinylated circRNA Pulldown Assay
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Cells (0.25 × 105) seeded in 6-well plates were transfected with biotinylated control or circRNA probe (100 nM) using Lipofectamine™ 2000. After 48 h, cells lysates (from 1 × 106 cells) were collected by using IP Lysis Buffer (Beyotime Biotechnology), and 10% of the lysates was employed as the input. The remaining lysates were incubated with M-280 streptavidin magnetic beads (Sigma-Aldrich) at 4 °C overnight. A magnetic bar was used to precipitate the magnetic beads. After 4 washes with a high-salt wash buffer, both the input and the precipitated samples from the pull-down were purified with TRIzol® reagent (Invitrogen). The enriched miRNA was then detected by reverse transcription-quantitative PCR (RT-qPCR) analysis.
7
RNA-Protein Interaction Purification
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As previously described 43 (link), the RNA affinity purification was performed. The 5'biotin-labeled PKM EI9 RNAs were synthesized by Genscript company (Nanjing, China) according to Chen et al.'s research 29 (link). 1 nmol biotin-labeled RNAs was bound with 50 μl Streptavidin-Agarose beads (Sigma, GE17-5113-01) to prepare RNA-immobilized beads. Furthermore, cells grown at 70-80% confluency were transfected with the indicated plasmid for 48h. Cells were lysed in IP lysis buffer (beyotime, P0013) adding 1× phosphatase inhibitor cocktail (Sigma-Aldrich & Roche). Nuclear pellets were collected with Nuclear and Cytoplasmic Protein Extraction Kit, and lysed by sonication. Nuclear lysates were incubated with RNA-immobilized beads at 30 °C for 30 min while rotating. After protein and RNA binding, proteins were eluded with using SDS lysis buffer (beyotime, P0013G). The samples were detected by Western blotting.
8
Immunoprecipitation of Fbxo22 and KDM5A Proteins
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To immunoprecipitate the exogenous proteins, the indicated plasmids (Flag-Fbxo22 and HA-KDM5A) were transfected into HEK293T cells. Cell lysis was carried out in IP lysis buffer (P0013, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Cell lysis buffer was obtained by centrifugation at 12,000 g for 20 min at 4 °C. The cell lysis buffer containing 200 μg protein was then incubated with anti-flag antibody (1:50, F3165, Sigma-Aldrich) or anti-HA antibody (1:50, #3724, Cell Signaling, Hercules, CA) for 4 h at 4 °C.
To immunoprecipitate endogenous proteins, the same method as above-described was performed. Antibodies of lysates, KDM5A (1:100, ab70892, Abcam, UK), and IgG (1:50, #3900, Cell Signaling), at a concentration of 1 μg/mg, were added to the cell lysate and incubated overnight at 4 °C. The antibody-protein complexes were then captured with protein A/G Sepharose microbeads (Santa Cruz, CA). The complexes were then subjected to immunoblotting with mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz) and mouse anti-KDM5A (1:1000, ab78322, Abcam).
To immunoprecipitate endogenous proteins, the same method as above-described was performed. Antibodies of lysates, KDM5A (1:100, ab70892, Abcam, UK), and IgG (1:50, #3900, Cell Signaling), at a concentration of 1 μg/mg, were added to the cell lysate and incubated overnight at 4 °C. The antibody-protein complexes were then captured with protein A/G Sepharose microbeads (Santa Cruz, CA). The complexes were then subjected to immunoblotting with mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz) and mouse anti-KDM5A (1:1000, ab78322, Abcam).
9
Evaluating UV-Induced DNA Damage
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Cells were seeded at a density of 3 × 105 cells per well in six-well plates. Ultraviolet (UV) irradiation (dose rate: 20 mJ/40 mJ/80 mJ, wavelength 365 nm, field: 20 × 20 cm) was used to irradiate A2780 and SKOV3 cells, while a well without UV irradiation was used as a control. Subsequently, the cells were cultured for 24 hours, collected, and lysed for total protein extraction using immunoprecipitation (IP) lysis buffer (Beyotime Biotechnology, Shanghai, China). Western blotting was used to measure the expression of NBS1, CyclinB, and other protein molecules, with pH2AX serving as an indicator of double-strand break repair.
10
Protein Purification and Interactome Analysis
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Cells transfected with GST-tag segments of HDAC5 plasmids were lysed with IP lysis buffer (Code No. P0013, Beyotime Biotechnology) on ice about 0.5 hour, followed by ultrasonication. GST fusion proteins were purified using GST fushion protein purified magnetic beads (Sorlabio Life Science). Then the purified proteins beads were incubated along with cell lysates overnight at 4°C. The beads were washed eight times on ice using ice-cold binding buffer (20 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L EDTA, 5% Glycerol, 1 mmol/L DTT, 1 mmol/L PMSF, 1 mmol/L Benzamidine) and proteins were eluted on ice using ice-cold IP lysis buffer and analyzed by Western blotting.
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