In brief, cells were lysed in IP buffer (Beyotime) containing 0.5 mM PMSF (Beyotime) and 1x phosphatase inhibitor (Roche). Cell lysate was centrifuged and supernatants are precleared with 10 μl protein A/G beads (Beyotime) at 4˚C for 1 h. Cell lysate was then incubated with antibodies at 4˚C overnight prior to addition of 20 μl protein A/G beads for 2 h at 4˚C. Immunoprecipitated proteins were extensively washed and heat denatured at 95˚C for 5 min. The protein samples were separated by SDS-PAGE and analyzed by Western blot. The following antibodies were used: anti-flag tag antibody (Cell signaling technology, #14793, 1:50), anti-NOTCH3 antibody (Cell signaling technology, #5276, 1:200), Normal IgG (Sigma-Aldrich, 12-371, 1:1000).
Protein a g beads
Manufactured by Beyotime
Sourced in China, United States
Protein A/G beads are a type of affinity chromatography resin used for the purification of antibodies. They contain a mixture of Protein A and Protein G, which are bacterial cell wall proteins that bind to the Fc region of immunoglobulins. These beads provide a simple and efficient method for the capture and isolation of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Complete cocktail, by Roche (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
St476, by Beyotime (2 mentions)
Sds loading buffer, by Beyotime (2 mentions)
Lumiglo chemiluminescent substrate system, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Leupeptin, by Beyotime (2 mentions)
Np 40 buffer, by Beyotime (2 mentions)
Ab172730, by Abcam (2 mentions)
Proteinase k, by Takara Bio (2 mentions)
Rnase, by Takara Bio (2 mentions)
Tom20 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti notch3 antibody, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Normal igg, by Merck Group (1 mentions)
Anti flag tag antibody, by Cell Signaling Technology (1 mentions)
Ip buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
52 protocols using protein a g beads
1
Immunoprecipitation of NOTCH3 Protein
2
Immunoprecipitation and Mass Spectrometry
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T24 cells cultured in a 10 cm dish were harvested and lysed with 1 mL RIPA buffer (Beyotime) containing 1% protease inhibitor (Sigma-Aldrich; Shanghai, China) on ice for 30 min. The supernatant was obtained by centrifugation and divided into treatment and control groups. 50 µL of prewashed protein A&G beads (Beyotime) and 20 µg HEK293-produced and purified Siglec9-Fc were added to the treatment group, and an equal amount of protein A & G beads were added to the control group. After mixing at 4 °C for 2 h, the beads were washed with PBS and eluted with 0.2 M glycine-HCl buffer (pH = 3.0) to get proteins; the pH of the solution was then adjusted to 7.0 using 1 M Tris-HCl buffer (pH = 8.5). IP proteins or 20 µg purified Siglec9-Fc were reduced using 10 mM DTT at 56 °C for 45 min and alkylated with 20 mM IAM in the dark for 30 min at room temperature. Sequencing grade trypsin (Shengxia; Beijing, China) was added to the solution at 1:40 (w/w), followed by incubation overnight at 37 °C. The peptides were desalted using C18 resin pipet tips (ZipTip, Millipore Corp, Billerica, MA, USA).
3
Bnip3 Interaction with TOM20 via Immunoprecipitation
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Immunoprecipitation (IP) was performed as described elsewhere [35 (link)]. Myocardial tissues were homogenized in RIPA buffer, and the protease inhibitor PMSF was added. The homogenates were centrifuged at 12,000 rpm for 20 min at 4°C, and 200 μL of the supernatant liquor was collected. Following the standard of 1 μg antibody per 500 μg protein, Tom20 antibody (Santa Cruz Biotechnology) was added for overnight incubation, and the negative control used IgG to replace the Tom20 antibody. Then, the homogenates were incubated with 40 μL of Protein A/G beads (Beyotime Biotech, Jiangsu, China) for 3 h at 4°C. Beads were washed five times in RIPA buffer then centrifuged at 2500 rpm for 5 min each time at 4°C, and the immunoprecipitated proteins were used to analyze the binding relationships between Bnip3 and TOM20 by the Western blotting method. Bnip3 expression of the TOM20-immunoprecipitated products was detected and was balanced by TOM20 blotting results.
4
SIRT1 and PGC-1α Interaction Assay
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HepG2 cells were treated with Cd and lysed with cell lysis buffer (P0013, Beyotime Company, Shanghai, China). Lysates were clarified by centrifugation at 12,000 g for 15 min and were used for immunoprecipitation. Typically, 2 μg of SIRT1 or PGC-1 alpha antibody was incubated with 500–1000 μg of cell lysate protein overnight at 4°C, then protein A/G beads (P2006 or P2009, Beyotime Company, Shanghai, China) were added and the mixture was incubated overnight at 4°C. Then, the beads were washed three times, solubilized in 40 μl 3xSDS sample buffer (7722, Cell Signaling Technology, MA), and analyzed by immunoblotting with antibodies against PGC-1 alpha or acetylated-lysine, respectively.
5
Protein Extraction and Co-Immunoprecipitation
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Cells were extracted with lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES, 1 mM PMSF, 1 mM DTT) and homogenized for 30 min at 4 °C. Protein extracts were centrifuged at 12 000 × g for 15 min at 4 °C, and then the supernatants containing total protein were collected. Equal amounts of protein were exposed to antibodies against control immunoglobulin G (1:100, Abcam, Cambridge, MA, USA, ab200699) or Nek7 (1:10, Abcam, ab133514), which was immobilized on protein A/G beads (Beyotime). After a 3 h incubation at 4 °C with gentle rotation, beads were washed extensively five times with lysis buffer, boiled and microcentrifuged. Proteins were detected with monoclonal antibodies against Nek7 (1:10 000, Abcam, ab133514), NLRP3 (1:1000, Proteintech, No. 19771-1-AP) by western blot.
6
Protein-Antibody Pulldown Assay
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Pull-down assays were performed using protein A/G beads purchased from Beyotime Technology (Jiangsu, China; Cat. P2012). We followed the standard protocol provided by the manufacturer with minor adjustments. Specifically, 2 μg antibody was used in each reaction. protein A/G beads were immediately added to the protein-antibody solution and incubated at 4 °C overnight. Beads were washed with 1× PBS (diluted from 10× PBS, ST476, Beyotime Technology) at least 3 times before western blotting (WB) analysis.
7
Immunoprecipitation and Western Blot
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Cell lysates from cells of interest were prepared in a protein cell lysis buffer (25 (link)). After clarification and concentration determination, an equal number of total proteins were incubated with specific antibodies overnight at 4 °C followed by incubation with Protein A + G beads (Beyotime Biotechnology) for 2 h at room temperature. After washing, the protein-containing beads were collected and boiled in 2× SDS loading dye before being subjected to IB assays.
8
Immunoprecipitation with Antibodies
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Cells were lysed on ice in RIPA buffer containing 1% Cocktail (Servicebio). 10% input was saved, and the cell lysate was incubated with IgG (Proteintech) or primary antibody (AURKB, MAD2L2) at 4 °C overnight on a rotator, followed by Protein A + G beads (Beyotime, Shanghai, China) for another 2h of incubation. The supernatant was taken from the beads after centrifugation. The beads were washed three times using the inhibitor-containing lysate, and then boiled with Sample Loading Buffer (Beyotime) at 95 °C for 10 min before SDS-PAGE and western blot.
9
Whole-Cell Lysate Immunoprecipitation Protocol
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The method of extracting the whole-cell lysate used for Co-IP assays and ubiquitination assays was consistent with the method mentioned in Western blot assays. The whole cell lysate was then incubated with the detection antibody overnight at 4 °C. The next day, 50 µl of protein A/G beads (Beyotime) were added into the protein samples and remixed by rotation for 6 h. Lastly, the samples were detected by 7.5% SDS–PAGE electrophoresis after centrifugation to remove the supernatant.
10
Immunoprecipitation Assay Protocol
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For the immunoprecipitation assays, whole-cell extracts were prepared after transfection, incubated overnight with the appropriate antibodies and subsequently incubated with Protein A/G beads (Beyotime). The beads were washed 5 times with low-salt lysis buffer, and the immunoprecipitates were eluted in 1 × SDS Loading Buffer (Beyotime) and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes (Bio-Rad) and incubated with the appropriate antibodies. The LumiGlo Chemiluminescent Substrate System (Thermo) was used for protein detection. All original western blotting blots are presented in Supplementary Figs 13 –27 .
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