Cells were collected and lysed in IP lysis buffer (150 mM NaCl, 50 mM Tris pH8, 1% Triton X-100, 1% NP-40) supplemented with protease and phosphatase inhibitors, incubated on ice for 20 min, and cleared by centrifugation at 12,000 rpm at 4 °C for 10 min. Total protein lysate (1 mg) was immunoprecipitated with antibody (anti-PIWIL2, HDAC3 or IgG as control antibodies) and incubated on a rotator overnight at 4 °C and followed by incubation with protein A + G agarose beads (Beyotime Biotechnology, Shanghai, China) for 2 h. Immune complexes were eluted from the agarose beads and analyzed by SDS-PAGE followed by immunoblot analysis.
Agarose beads
Manufactured by Beyotime
Sourced in China
Agarose beads are a type of porous, cross-linked polymer material commonly used in laboratory applications. They are composed of agarose, a polysaccharide derived from red algae. Agarose beads possess a high degree of chemical and physical stability, making them suitable for a variety of separation and purification techniques.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g agarose bead, by Beyotime (5 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Protein a agarose, by Beyotime (2 mentions)
Ab33168, by Abcam (1 mentions)
Immunoprecipitation buffer, by Beyotime (1 mentions)
Ab18203, by Abcam (1 mentions)
Protein a agarose beads, by Beyotime (1 mentions)
Protein g agarose beads, by Beyotime (1 mentions)
Antibody against beclin 1, by Cell Signaling Technology (1 mentions)
Anti ha, by Proteintech (1 mentions)
11 protocols using agarose beads
1
Immunoprecipitation and Immunoblot Analysis
2
Immunoprecipitation and Western Blotting
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Cells were lysed in immunoprecipitation buffer (Beyotime Biotechnology, Shanghai, China), and equal amounts of 500 μg protein were incubated with specific antibodies against PlexinB1 (ab90087, Abcam), N‐cadherin (ab18203, Abcam), and VE‐cadherin (ab33168, Abcam) overnight at 4°C with gentle rotation. 40 μl Protein A&G Agarose beads (Beyotime Biotechnology) were then added and incubated for another 2 h. Agarose beads were precipitated to bottom by transient centrifugation at 4°C and then washed three times with the lysis buffer. The complexes were eluted from the beads by heating samples in loading buffer with SDS and then used for Western blotting.
3
Protein Interaction Analysis via Co-IP and Immunoblotting
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To investigate the interactions of the proteins, coimmunoprecipitation and immunoblotting were performed as follows. HEK293T cells were lysed in 1 mL of lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, and 2 mM Na2EDTA). After the lysate was collected by centrifugation at 12000 × g for 10 min, the lysate proteins were incubated overnight at 4 °C with 0.5 μg of the indicated antibody; 30 μl of Protein A+G agarose beads (Beyotime, China) was then added to each immunoprecipitation reaction for another 6 h. The agarose beads were then washed three times with 1 mL of lysis buffer. The precipitates were subjected to immunoprecipitation with an anti-Flag antibody, and the captured proteins were detected by immunoblotting with the indicated antibodies.
4
Protein Interaction Profiling in HEK293 T Cells
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To investigate the interactions between proteins, HEK293 T cells were lysed in immunoprecipitation lysis buffer (RIPA). After the lysates were incubated for 1 h at 37 °C with RNase and DNAse, the lysate proteins were incubated overnight at 4 °C with the indicated antibodies. Protein A + G agarose beads (30 μl; Beyotime) were then added to each immunoprecipitation reaction for another 6 h. The agarose beads were then washed three times and the captured proteins were resolved on 8%–12% SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting.
5
Co-IP Assay for Protein-Protein Interactions
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For co-IP experiments, cells were collected and lysed with pre-cooled IP lysis buffer (cat. no. P0013) containing protease inhibitors (cat. no. P1046; both from Beyotime Institute of Biotechnology) for 10 min on ice. The supernatant was collected after centrifugation at 13,000 x g for 10 min at 4°C. Subsequently, the lysate (0.5 mg per IP reaction) was supplemented with 1 µg SIK (cat. no. 6919; dilution, 1:50; Cell Signaling Technology, Inc.) or TRIM28 antibody (cat. no. ab109545; dilution, 1:30; Abcam) and 10 µl protein A agarose beads (Beyotime Institute of Biotechnology) followed by gentle rotation at 4°C for 4 h. Following centrifugation at 1,000 x g for 3 min at 4°C, the supernatant was carefully removed and the agarose beads in the bottom of the tube were washed with lysis buffer. The pellets were then dissolved in 15 µl 2× SDS sample buffer and boiled for 5 min, followed by western blot analysis.
6
Immunoprecipitation of Protein Interactions
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To investigate the interactions between proteins, HEK293T cells or MARC-145 cells were lysed in immunoprecipitation lysis buffer (RIPA). After the lysate was clarified by centrifugation at 12,000 × g for 10 min, the lysate proteins were incubated overnight at 4°C with the indicated antibodies. Protein A + G agarose beads (30 µl; Beyotime) were then added to each immunoprecipitation reaction for another 6 h. The agarose beads were then washed three times and the captured proteins resolved on 8–12% SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting.
7
Protein-Protein Interaction Detection
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To detect protein–protein interactions, cells were lysed in 500 μl co-IP buffer supplemented with a cocktail of proteinase inhibitors, phosphatase inhibitors, and RNase inhibitors. The lysates were added to the same IgG species used for immunoprecipitation as the normal IgG and agarose beads (Beyotime, Shanghai, China) and shaken slowly at 4 °C for 30 min to 2 h. The supernatant is centrifuged at 1000 g for 5 min and used for immunoprecipitation with agarose beads, which were pre-incubated with the corresponding antibodies. After incubation at 4 °C overnight, beads were washed 5 times with PBS. SDS sample buffer was added to the agarose beads, and immunoprecipitates were used for western blot analysis.
8
Beclin-1 Immunoprecipitation Assay
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MSCs were quickly harvested and homogenized on ice in modified RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (vol/vol) Triton X-100, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 1 mM EDTA, 50 mM N-ethylmaleimide, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF and 1 μg ml−1 each of aprotinin, leupeptin and pepstatin. The cell extracts (~200 μg of total protein) were incubated with an antibody against Beclin-1 (2.5 μl, Cell Signaling Technology) at 4 °C overnight. Then, protein-G agarose beads (40 μl, Beyotime Biotechnology, Shanghai, China) were added, and the mixture was incubated at 4 °C for 3 h. The agarose beads were collected, washed, and resuspended in 60 μl of sample buffer containing 50 mM Tris-HCl, pH 7.6, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 10 mM DTT and 0.2% bromophenol blue. Afterwards, the samples were boiled for 5 min and analyzed via western blot.
9
Proteomic Profiling of FBXL11 Interactome
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Total protein in OC cells was extracted by a mild RIPA lysis reagent (Thermo Fisher Scientific, USA) containing 1% PMSF and 1% phosphorylase inhibitor (Boster, Wuhan, China). Primary anti-FBXL11 (1 μg), anti-HA (1 μg; Cat No. 51064-2-AP, Proteintech, Wuhan, China), and anti-Flag antibodies (1 μg; Cat No. 80010-1-RR, Proteintech, Wuhan, China) were used to bind FBXL11, HA-conjunct, and Flag-conjunct proteins at 4 °C overnight. Agarose bead gel (Beyotime, Suzhou, China) was used to enrich the antigen-antibody complex at 4 °C for 3 h. Then, after washing PBS for thrice, agarose beads were obtained using centrifugation. The agarose bead gel was mixed with 2× loading buffer and performed degeneration at 100 °C for 10 min. Proteins interacting with FBXL11 were identified via LC/MS and visualized via silver staining using a rapid silver stain kit (Beyotime, Suzhou, China) according to the manufacturer’s instructions. Finally, the expression of target proteins was verified by western blotting.
10
Protein Immunoprecipitation and Western Blot
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Cells were treated with cell lysis buffer (Beyotime, China) to obtain cell lysates and retain the input material. agarose beads (Protein A Agarose, Beyotime, China) were incubated with an antibody at 4°C for 2 h to ensure that the agarose beads bound to the antibody. agarose beads bound to the antibody were added to the cell lysate and shaken at 4°C overnight. The sample protein was obtained by repeated elution of nonspeci c binding proteins. The sample was mixed with SDS-PAGE loading buffer and heated to 100°C for 10 min. The results were detected by western blot. IPKine™ HRP, Mouse Anti-Rabbit IgG LCS (Abbkine, China) was used as the secondary antibody.
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