IgG, HA, and GSK3β were added into lysis buffer gained from cells and incubated overnight at 4℃ with shaking. Then we mixed mix protein A/G PLUS‐agarose (Beyotime) and cell lysates and incubated with shaking for 2 h. Lysis buffer was used to wash the agarose beads for 3 times. The final cell lysates were separated by Western blotting. Total proteins were obtained using RIPA buffer (Beyotime) and separated on 8%–12% SDS‐PAGE. According to previous study,27 the PVDF membranes were probed by antibodies. The antibodies were listed as follow: β‐catenin (66379–1‐Ig, Proteintech, 1:1,000), Phospho‐β‐catenin (#9561, Cell Signaling Technology, 1:1,000), GSK3β (67329–1‐Ig, Proteintech, 1:1,000), HA tag (#5017, Cell Signaling Technology, 1:1,000), Vimentin (ab92547, Abcam, 1:1,000), E‐cadherin (ab40772, Abcam, 1:1,000), and β‐actin (AA128, Beyotime, 1:1,000). The expression levels were developed under enhanced chemiluminescence (ECL, Millipore).
Protein a g plus agarose
Manufactured by Beyotime
Sourced in China
Protein A/G plus agarose is a type of lab equipment used for the purification and isolation of antibodies. It consists of protein A and protein G, which are immobilized on agarose beads. Protein A and protein G are bacterial proteins that have a high affinity for the Fc region of various immunoglobulin classes, allowing for the efficient capture and separation of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Phospho β catenin, by Cell Signaling Technology (1 mentions)
Ab40772, by Abcam (1 mentions)
Aa128, by Beyotime (1 mentions)
β catenin, by Proteintech (1 mentions)
Ab92547, by Abcam (1 mentions)
Gsk3β, by Proteintech (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Ip lysis buffer, by Merck Group (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Hiscript 3 rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Luciferase reporter gene kit, by Beyotime (1 mentions)
Ecl advance reagent, by Beyotime (1 mentions)
Aceq qpcr sybr green master mix without rox, by Vazyme (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Normal igg, by Beyotime (1 mentions)
8 protocols using protein a g plus agarose
1
Co-Immunoprecipitation and Western Blot Analysis
2
Co-Immunoprecipitation Assay for Protein-Protein Interactions
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Co-IP assays were performed as previously described (113 (link), 114 (link)). Hone1-EBV cells were treated with TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce EBV lytic infection, or HEK293T cells were transfected with the indicated plasmid(s) for 24 h. After EBV infection or plasmid transfection, cells were collected and lysed in RIPA lysis buffer (Beyotime) on ice for 30 min. The supernatant was subsequently incubated with 3 μg of the indicated Ab(s) or nonspecific IgG at 4°C overnight. A 1:1 slurry of protein A/G plus agarose (Beyotime) was added for 4 h. The bead-antibody-protein complexes were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB.
3
Ubiquitin Regulation of NOTCH1 Signaling
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The cell was transfected with ubiquitin plasmid and treated with 10 μM MG132 (Selleck) for 6 h. Total protein was then isolated using RIPA lysis buffer. Approximately 500 μg/250 μl protein per sample was incubated with the NOTCH1 antibody at 4 °C overnight and followed by incubated with Protein A/G Plus-Agarose (Beyotime) at 4 °C overnight. After being washed in PBS and centrifuged, the ubiquitin level in the precipitation was detected by immunoblotting.
4
Western Blotting and Co-Immunoprecipitation Assays
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For western blotting assay, cells were lysed by ice-cold RIPA lysis buffer (Beyotime, Shanghai) for 1 h. The supernatant was harvested for further measuring its concentration by BSA reagents (Beyotime, Shanghai) in 560 nm wavelength. The amount of a particular supernatant was mixed up and boiled with loading buffer (Beyotime, Shanghai), and was followed by SDS-electrophoresis, primary antibody incubation, HPR-linked secondary antibody incubation, and exposure. The detail was performed as previously described [27 (link)]. The results of the western blot were analyzed by using ImageJ software. And the quantifications were calculated with methods of normalized vs. the tubulin loading control or the experimental group vs. the control group.
For Co-IP assay, cells were lysed in ice-cold IP lysis buffer (Sigma, USA) and then incubated on a rocker with indicated antibody as well as IgG at 4 °C overnight. After incubation with Protein A/G PLUS-Agarose (Beyotime, Shanghai). The loading buffer was added and boiled in the water bath kettle. The supernatant was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
For Co-IP assay, cells were lysed in ice-cold IP lysis buffer (Sigma, USA) and then incubated on a rocker with indicated antibody as well as IgG at 4 °C overnight. After incubation with Protein A/G PLUS-Agarose (Beyotime, Shanghai). The loading buffer was added and boiled in the water bath kettle. The supernatant was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
5
Synthesis and Characterization of JAC1 Proteins
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JAC1 and Biotin-JAC1 were synthesized by the laboratory-self, with the purity of > 98%. Primary antibodies information is listed in Supplementary Table 1 . ECL advance reagent, CCK-8 Kit (C0038), Nucleoplasmic protein isolation Kit (P0027), Luciferase reporter gene Kit (RG027), Protein A/G Plus-Agarose (P2055) were purchased from Beyotime (Shanghai, China). Annexin V-FITC/PI apoptosis detection Kit (A211-02), HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01), AceQ qPCR SYBR Green Master Mix (without ROX) were obtained from Vazyme Biotech Co (Nanjing, China). CHX (761982), MG132 (C2211) were obtained from Sigma (St. Louis, MO).
6
In Vitro Co-Immunoprecipitation of FLAG-Tagged Proteins
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For in vitro co-immunoprecipitation (co-IP) analysis, FLAG-tagged proteins were produced in HTR-8/Svneo and JAR cells transfected with LV-PADI6. Total cell lysates were incubated overnight at 4°C with anti-YAP1 antibodies (Proteintech, Wuhan, China), anti-FLAG antibodies (Sigma, MO, USA), or normal IgG (Beyotime, Shanghai, China) as a control. Antibody-antigen complexes were precleared with Protein A/G PLUS-Agarose (Beyotime, Shanghai, China). After several washes, samples were boiled and analyzed by immunoblot.
7
TGEV-Infected Cell Lysate Immunoprecipitation
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The lysate of ST cells infected with TGEV for 24 h was prepared with RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate) containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (1 mM). After centrifugation at 12,000 ×
g for 15 min, lysate supernatant was pretreated with protein A/G plus-agarose (Beyotime) for 30 min at 4 °C to eliminate non-specific binding to the agarose beads. The lysate supernatant (500 μg) was incubated with 1 μg of rabbit pAb to EF1A for overnight at 4 °C. Then, 20 μL resuspended protein A/G plus-agarose was added to this mixture and incubated at 4 °C on a rocker platform for 2 h. After washing four times with lysis buffer, the isolated immunoprecipitated proteins were then analyzed by western blotting using mAb to N protein of TGEV and rabbit pAb to EF1A. The lysate of TGEV mock-infected ST cells was used as a control.
g for 15 min, lysate supernatant was pretreated with protein A/G plus-agarose (Beyotime) for 30 min at 4 °C to eliminate non-specific binding to the agarose beads. The lysate supernatant (500 μg) was incubated with 1 μg of rabbit pAb to EF1A for overnight at 4 °C. Then, 20 μL resuspended protein A/G plus-agarose was added to this mixture and incubated at 4 °C on a rocker platform for 2 h. After washing four times with lysis buffer, the isolated immunoprecipitated proteins were then analyzed by western blotting using mAb to N protein of TGEV and rabbit pAb to EF1A. The lysate of TGEV mock-infected ST cells was used as a control.
8
Co-immunoprecipitation of NCOA4 Protein
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Following the corresponding pretreatment in the indicated groups, the concentration of protein in MLE-12 cells was assessed by the BCA protein (Beyotime, Shanghai, China) assay kit. MLE-12 cells were lysed in mixed buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L NaF, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, and protease inhibitor cocktail). The protein supernatant was hatched with 1–2 μg rabbit polyclonal IgG control antibody and 25 μl resuspended volume of protein A/G plus agarose (Beyotime, P2055) for 1 h. Then, the protein supernatant was incubated with 1 μl anti-NCOA4 (Affinity Biosciences, DF4255, 1:1,000) for 24 h at 4°C. The cells were cultured again lasting 2 h. The immunoprecipitation buffer was washed several times, and 50 μl of SDS-PAGE loading buffer was added and then denatured. Finally, the co-IP assay was performed.
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