HEK293 cells were seeded in 25-cm2 culture flasks overnight and transfected with 5 μg of plasmid DNA. At 24 h, medium was removed and the cell monolayer was washed with PBS (pH 7.4, Gibco) and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate] (Beyotime) containing protease inhibitor cocktail (CWBio) on a rocker platform at 4°C for 30 min. The cell lysate was centrifuged at 12,000 rpm at 4°C for 15 min, and the supernatant was collected. Eighty microliters of lysate supernatant was mixed with 20 µl of 5× SDS sample buffer and incubated with 30 µl of Protein A/G Agarose Resin (Yeasen) to remove background. Then, the corresponding primary antibody was added and incubated at 4°C overnight with constant agitation (dilution ratio according to antibody specification). One hundred microliters of 50% Protein A/G Agarose Resin (Yeasen) was added. After incubation at room temperature for 1 h, immunoprecipitated proteins were collected by centrifugation at 2,500 g for 3 min at 4°C, washed three times with ice-cold PBS, resuspended in 80 µl of 2× SDS-PAGE sample loading buffer, and subjected to 12% SDS-PAGE and Western blotting.
Protein a g agarose resin
Manufactured by Yeasen
Sourced in China
Protein A/G Agarose Resin is a laboratory product used for the purification of antibodies from various samples. It consists of recombinant Protein A and Protein G, which are immobilized on an agarose matrix. The resin can be used to selectively bind and capture antibodies, enabling their isolation and purification for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Np 40 lysis buffer, by Beyotime (3 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Human anti ago2 antibody, by Cell Signaling Technology (1 mentions)
Tyrosine phosphatase assay kit, by Promega (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Anti ha, by Proteintech (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Anti flag m2 beads, by Selleck Chemicals (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti flag antibody, by Abmart (1 mentions)
Anti gfp antibody, by Abmart (1 mentions)
10 protocols using protein a g agarose resin
1
Immunoprecipitation and Western Blotting Protocol
2
miR-873 regulation of PD-L1 3'UTR
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RIP assay was performed using the Protein A/G Agarose Resin (YEASEN, China) following the manufacturer's protocol. The detailed procedure was described in our previous studies [28 (link),29 (link)]. MCF-7 cells were transfected with pcDNA3.1(+) PD-L1 3′UTR (WT) and pcDNA3.1(+) PD-L1 3′UTR (MUT), and lysed by NP-40 lysis buffer (Beyotime, China). Ago2-RNA complex was dissociated from the beads. qRT-PCR was performed to detect miR-873 level.
3
RETSAT Co-Immunoprecipitation Protocol
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Co-immunoprecipitation (Co-IP) was performed as described previously [36 (link)]. Specifically, PANC-1 cells were harvested and washed twice with ice-cold PBS and lysed with 1× RIPA lysis buffer (Beyotime, P0013D) containing complete EDTA-free (Roche) inhibitors. Immunoprecipitation with RETSAT antibody performed on and with Lysates were digested by 10 units/mL DNase I (New England Biolabs, M0303), and incubated with anti-RETSAT primary antibody overnight. Isotype IgG were used as negative controls. Immunoprecipitation was carried out using protein A/G Agarose Resin (Yeasen, 36403ES08) according to the manufacturers’ protocol. After pulling down and wash, proteins were fractionated by SDS-PAGE gel for immunoblotting.
4
RIP Assay for miR-375 Targeting
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RIP assay was performed using the Protein A/G Agarose Resin (YEASEN, China) following the manufacturer’s protocol. For RIP assay, SLC7A11 3′UTR sequences containing miR-375 binding sites or mutated binding sites were inserted into pcDNA3.1 (+) plasmid, named as SLC7A11 3′UTR (WT) or SLC7A11 3′UTR (MUT), respectively. SGC-7901 and BGC-823 cells were transfected with SLC7A11 3′UTR (WT) and SLC7A11 3′UTR (MUT). After 24 h, cells were extracted and lysed by NP-40 lysis buffer (Beyotime, China). After that, 100 μL lysate was incubated with NP-40 buffer containing Protein A/G Agarose Resin bound with human anti-Ago2 antibody (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. Agarose resin was isolated by centrifugation and digested with protease K (YEASEN) to isolate Ago2-RNA complex from the beads. qRT-PCR was performed to detect miR-375 level [44 (link)].
5
Measuring SHP-2 Phosphatase Activity
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After treatment of TA, U2OS or xenograft tumor tissue protein extract were incubated with anti-SHP-2 antibody (Ab) in immunoprecipitation (IP) buffer (20 mM of Tris-HCl, 150 mM of NaCl, 1 mM of ethylenediaminetetraacetic acid, 1 mM DTT, 1% beta-mercaptoethanol, 1% TritonX-100, 1 mM MgCl2 and 0.1% BSA, 1% Protease inhibitor, pH 7.5) overnight. Protein A/G-Agarose Resin (Yeasen, Shanghai, China) was added to each group, followed by incubation for 3 h at 4 °C with rotation. After centrifugation, SHP-2 PTP activity was measured using the Tyrosine Phosphatase Assay Kit (Promega, Madison, WI, USA) as specified by its manufacturer. The purified recombinant SHP-2 proteins (Novus Biologicals, Littleton, CO, USA) were preincubated with different concentrations of TA in the reaction mixture. The detection method of purified SHP-2 PTP activity was as same as the IP samples.
6
Immunoprecipitation of NLRP3 Protein
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Proteins were extracted from cultured cardiomyocytes or cardiac tissue and incubated with primary antibodies at 4°C overnight. The antibodies used in this study were as follows: anti-IgG (Cell Signaling Technology, #3900, 1:200), anti-NLRP3 (Cell Signaling Technology, #15101, 1:200, or Abcam, #ab263899, 1:30), and anti-HA (Proteintech, Cat No. 51064-2-AP, 1:100). The mixture was then incubated with 30 μL of Protein A/G Agarose Resin (YEASEN, #36403ES03) for 2 hours with rotation. After eluting the surface nonspecific binding, samples were obtained from the resin–antibody complexes and subjected to immunoblot.
7
Protein Interaction Assay for SEMA3A-NRP1
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The binding relationship between SEMA3A and NRP-1 was examined through IP assay. The antibody was mixed with cell lysate containing the target protein and incubated at room temperature for 60 min. Protein A/G Agarose Resin (Yeasen, Shanghai, China) was added into a 2 mL tube and centrifuged at 500 rpm for 1 min, and the supernatant was discarded. Following resuspension, the antigen-antibody mixture was added to the resin, sufficiently mixed and centrifuged to collect the supernatant. After elution of the sample, the precipitate is detected.
8
Musclin-Fc Protein Purification from Expi293F Cells
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Expi293F cells were transfected with pcDNA3.0-Musclin-Fc using PEI (Polysciences, 23966-2). At 24 h after transfection, 1 M sodium butyrate (1:1000) was added to suppress cell proliferation, followed by incubation for another 4–5 days. The residual cells and debris in the collected media were removed by centrifuging at 450 g (5 min) and 12,000 g (5 min), respectively. Musclin-Fc was purified with protein A/G agarose resin (YEASEN, 36403ES08), filtered with a 0.22 μm filter (Millipore), and stored at −80 °C for future use.
9
FLAG-tagged Protein Immunoprecipitation
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Control cells or cells transfected with expression plasmids were lysed in lysis buffer (150 mM sodium chloride, 50 mM Tris, pH 7.3, 0.25 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.2% sodium fluoride, 0.1% sodium orthovanadate, and a mixture of protease inhibitors from Roche Applied Science). Lysates were immunoprecipitated (IP) with anti-FLAG M2 beads (Bimake, B23102) or protein A/G agarose resin (YEASEN, 36403ES25). Samples were run in SDS/PAGE gels and analyzed by Western blotting with anti-FLAG or indicated antibodies.
10
Co-Immunoprecipitation Assay for Protein Interactions
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Co-IP assays were performed as previous description (Liu et al., 2010) , with minor modification. Briefly, these constructs were transiently expressed in 4-week-old N. benthamiana leaves, respectively, with the agroinfiltration expression system. Proteins were extracted with the NP40 lysis buffer (P0013F; Beyotime Biotechnology, Jiangsu, China). Corresponding antibodies were added to the cell extracts, and protease inhibitor cocktail and MG132 were also added to prevent protein degradation. The mixtures were kept shaking gently at 4°C for 3 h. The immunocomplex was captured by adding 50-μL ml -1 protein A/G agarose resin (Yeasen) and shaking for another 3 h. The agarose beads were recovered by centrifugation at 14,000g for 5 s and washed with cold PBS for three times. The precipitated samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected with anti-GFP antibody (Abmart, Shanghai, China; M20004) and anti-FLAG antibody (Abmart; M20008), respectively.
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