This two group cells (light and heavy) were harvested and lysed respectively in IP Lysis Buffer (Thermo, USA) (25 mM Tris•HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). After the protein concentration being normalized, equal protein amounts of each cell lysate were mix. The equally mixed sample (2 mg) were incubated with 10 μg rabbit anti-Flag polyclonal antibody (MBL,USA) in 1 ml IP Lysis Buffer overnight at 4 °C, and then were precipitated with 20 μl Protein A/G Plus-agarose (Roche, Switzerland). The immunoprecipitates were separated by 12% SDS–polyacrylamide gel electrophoresis. After being stained with Coomassie Brilliant Blue G-250, each lane of the gel was cut into 10 gel slices (0.5 cm × 0.5 cm) and then analyzed by LC-MS/MS at National Center of Biomedical Analysis (China).
Protein a g plus agarose
Manufactured by Roche
Sourced in Switzerland
Protein A/G Plus-agarose is a chromatography resin consisting of recombinant Protein A and Protein G covalently coupled to agarose beads. This resin is designed for the purification of antibodies from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ip lysis buffer, by Thermo Fisher Scientific (3 mentions)
Protease and phosphatase inhibitor, by Roche (3 mentions)
Ip lysis buffer, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti gst, by Proteintech (1 mentions)
Anti gfp, by Proteintech (1 mentions)
Anti gap43, by Merck Group (1 mentions)
Anti gapdh, by Bioworld Technology (1 mentions)
Anti active caspase 3, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Pepstatin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
4 protocols using protein a g plus agarose
1
Immunoprecipitation and Mass Spectrometry Analysis
2
Immunoprecipitation and Immunoblotting Protocol
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Cells were harvested and lysed respectively in IP Lysis Buffer (Thermo, USA) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). The protein samples were incubated with indicated antibody in 1 mL IP Lysis Buffer overnight at 4 °C, and then were precipitated with 20 μL Protein A/G Plus-agarose (Roche, Switzerland). After a brief centrifugation, the pellet was washed 3 times with IP Lysis Buffer. The lysates and immunoprecipitates were analyzed by immunoblotting.
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .
Immunoblotting was performed using indicated primary antibodies: anti-MAGE-G1 (B-Bridge, USA), anti-GFP (Proteintech, USA), anti-GAP43 (Sigma-Aldrich, USA), anti-Neuron-specific III β-tubulin (Bioworld, China), anti-GAPDH (Bioworld, China), anti-active Caspase3 (Sigma-Aldrich, USA), anti-FSCN1 (Sigma-Aldrich, USA) and anti-VIME (Sigma-Aldrich, USA), anti-GST (Proteintech, USA), anti-Flag (MBL, USA). Detailed information of immunoblotting analysis was previously described42 .
3
Immunoprecipitation and Western Blotting Protocol
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Cells were harvested at 48 h post-transfection and lysed respectively in IP Lysis Buffer (Thermo, Waltham, MA, USA) (25 mM Tris·HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). After the protein concentration of each sample in triplicate was determined using the BCA Protein Assay Kit (Thermo, Waltham, MA, USA), the sample (1 mg) were incubated with 3 µg rabbit anti-Flag polyclonal antibody (MBL, Woburn, MA, USA) or 3 µg rabbit anti-AFAP-120 polyclonal antibody in 1 mL IP Lysis Buffer for 8 h at 4 °C, and the immune complexes were precipitated with 20 µL Protein A/G Plus-agarose (Roche, Basel, Switzerland). The immunoprecipitates were then separated by 12% SDS–polyacrylamide gel electrophoresis.
4
Experimental Reagents and Materials
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RPMI-1640 medium, Dulbecco's modified Eagle medium, trypsin and TRIzol were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Puromycin and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc.). As2O3 was provided by Beijing Shuanglu Pharmaceutical Co., Ltd. (Beijing, China). Radioimmunoprecipitation assay lysates were purchased from Beyotime Institute of Biotechnology (Haimen, China). ATRA, phenylmethylsulfonyl fluoride, aprotinin, leupeptin and pepstatin were acquired from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Protein A/G PLUS-Agarose was obtained from Roche Diagnostics (Indianapolis, IN, USA). Anti-XBP1 antibody (cat. no. H00007494-D01) and anti-SUMO-1 antibody (cat. no. AJ1746a) were purchased from Abnova (Taipei City, Taiwan) and Abgent Biotech Co., Ltd. (Suzhou, China), respectively. The FITC Annexin V Apoptosis Detection kit and the anti-cluster of differentiation (CD) 11b antibody (cat. no. C09-550019) were commercially available from BD Pharmingen (San Diego, CA, USA). The PrimeScript RT reagent kit and SYBR Green PCR Master Mix were commercially available from Takara Bio, Inc. (Otsu, Japan).
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