Mouse antibodies against Myc-tag (M185-3L/M047-3, MBL, Nagoya, Japan), rabbit antibody against GFP-tag (D110008, BBI, Shanghai, China) and sheep antibody against Tubulin (ATN02, Cytoskeleton, Denver, United States) were subjected to Western blot according to the manufacturer’s protocol. For IP, HEK-293T cells transfected with the desired plasmids by using Attractene Transfection Reagent (301005, QIAGEN, Hilden, Germany), according to the fast-forward protocol, were lysed in IP buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF and Roche complete EDTA free protease inhibitor cocktail). After quantification by using BCA Protein Assay Kit (P0012, Beyotime, Beijing, China), lysates were precleared with Protein G Agarose (P2009, Beyotime, Beijing, China) and then immunoprecipitated with mouse anti-Myc antibodies. After washing five times, the precipitates were resuspended in the 5XSDS loading dye, boiled for 5 min, and run on a 12.5% SDS-PAGE gel followed by Western blot analysis. Immunoreactive bands were detected by Typhoon laser scanners (FLA-9500, GE, United States).
Protein g agarose
Manufactured by Beyotime
Sourced in China
Protein G agarose is a chromatography resin used for the purification of antibodies and other proteins. It consists of recombinant Protein G covalently coupled to agarose beads. Protein G has a high affinity for the Fc region of immunoglobulins, allowing for the selective capture and isolation of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Protein a agarose, by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Attractene transfection reagent, by Qiagen (1 mentions)
Atn02, by Cytoskeleton (1 mentions)
Typhoon laser scanner, by GE Healthcare (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Freund s incomplete adjuvant fia, by Merck Group (1 mentions)
Freund s complete adjuvant fca, by Merck Group (1 mentions)
Phosphatase inhibitor, by Active Motif (1 mentions)
Ab28147, by Abcam (1 mentions)
Ab198394, by Abcam (1 mentions)
Veriblot ip detection reagent, by Abcam (1 mentions)
Sc 8017, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Peg 1500, by Merck Group (1 mentions)
Amersham imager 600 imaging system, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Beyotime (1 mentions)
16 protocols using protein g agarose
1
Myc-tag Antibody Western Blot and IP
2
Polyclonal Antibody Production against GDCI3 i-antigen
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The purified recombinant GDCI3 i-antigen was emulsified with Freund’s Complete Adjuvant (FCA) (Sigma, USA) and 1 mg protein injected into New Zealand white rabbits (~1.5 kg). Animals were then boosted with 0.5 mg of purified antigen in Freund’s Incomplete Adjuvant (FIA) (Sigma, USA) on two separate occasions. Serum were then prepared and polyclonal antibody (pAb) titers determined by Enzyme-linked immunosorbent assay (ELISA) using recombinant GDCI3 i-antigen. Rabbit IgG were purified from rabbit antiserum using protein G agarose (Beyotime, Haimen, Jiangsu, China) according to the instructions of the manufacturer. Antibody specificity was determined by Western blotting using recombinant GDCI3 i-antigen and total protein from C. irritans theronts, trophonts, or tomonts as the antigen.
3
Ubiquitination Analysis of SLC7A11
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CO-IP analysis was performed to detect the ubiquitination level of SLC7A11. Total protein was extracted and quantified according to the method described in Sect. 2.6. 5 µL Protein A agarose and 5µL Protein G agarose (Beyotime, China) were added to the cell lysate and incubated at 4℃ for 1 h. Then, after centrifuging at 12,000 g, 4℃ for 1 min, 2 µg antibody, and the obtained supernatants were incubated overnight at 4℃, while using non-specific immune homologous IgG antibody as the control. The next day, the sediment was cleaned using 0.5 ml 1× wash buffer five times. The sediment was suspended in 30 µL of 1×SDS-PAGE electrophoresis sample buffer and then centrifuged for 30 s to allow the beads and liquid attached to the tube wall to reach the bottom of the tube. Then, The sample was placed at 100℃ for 5 min and centrifuged instantaneously at 14,000 g for 1 min. Finally, the collected supernatant was used for CO-IP. Western blot analysis was performed using the SLC7A11 antibody, and ubiquitination of SLC7A11 was detected using an anti-Ub antibody (Proteintech, China).
4
Immunoprecipitation and Western Blot Analysis
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Cell lysates were prepared using RIPA Buffer (Boston BioProducts) supplemented with protease inhibitors (Sigma) and phosphatase inhibitor (Active Motif). For immuoprecipitation experiments, cell lysates were incubated with desired antibodies in Tris buffered saline buffer at 4 °C overnight, then protein G agarose (Beyotime Biotechnology) was added to each sample for another 2 h incubation, followed by five times wash with PBS solution and boiling for IB. To avoid the interference from denatured IgG heavy chain, VeriBlot IP Detection Reagent (ab131366, Abcam) was used for IP detection. The antibodies against ARV7 (ab198394), Bcl-2 (ab59348), MCL-1 (ab28147) and Bim (ab15184) were obtained from Abcam while antibodies against cleaved-PARP (5625), Bid (2002), UBE2C (14234), p-H3(ser10) (9701), BubR1 (4116), and Cdc20 (14866) were purchased from Cell Signaling Technology, and the antibodies against GFP (sc-9996), p53 (sc-126), Mad-2 (sc-47747), and ubiquitin (sc-8017) were obtained from Santa Cruz. Proteintech is the provider of antibodies against β-actin.
5
Immunoprecipitation of Laminin Subunits
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All subsequent procedures were performed at 4°C, unless otherwise stated. A total of 1 ml HLE B-3 cell culture supernatant was incubated overnight on a shaker with protein G agarose (Beyotime Institute of Biotechnology) and anti-LMα4 rabbit pAb or anti-LMγ1 rabbit pAb. The same concentrations of normal rabbit IgG were used as an isotype control for IP. Precipitates, collected by centrifugation at 3,000 × g at 4°C for 2 min, were washed three times with PBS containing 0.5% Triton X-100, and were eluted from protein G agarose by boiling with 4X sample buffer for 2 min. Proteins in the supernatant were separated by SDS-PAGE and detected by western blotting, as aforementioned.
6
Receptor EDAR Interaction with EDA1
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Receptor EDAR: Fc (~500 μg in 200–500 μl of cell lysates) was added to 20–30 μl protein G agarose (Beyotime Institute of Biotechnology, Beijing, China), mixed with EDA1 proteins (~1 mg in 200–500 μl of cell supernatants) and incubated on a rotating wheel overnight at 4°C. protein G agarose was washed three times with 1 ml PBS after centrifugation at 3000 rpm for 5 min at 4°C. An equal volume of 2× sodium dodecyl sulfate (SDS) loading buffer was added to protein G agarose for resuspension and boiled for 10 min. Samples were analyzed by western blot with an anti‐EDA antibody (Abcam, Shanghai, China) and an anti‐Fc antibody (Abcam, Shanghai, China) for western blot analysis. Each experiment was repeated three times.
7
Immunoprecipitation and Western Blot Analysis
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A2780 cells (1.2 × 106 cells per dish) and ID8 cells (6 × 105 cells per dish) were seeded into 10 cm dishes and treated with FCCP and/or cisplatin. Cells were lysed in NP-40 buffer with 1× protease inhibitor. Lysates were shaken slowly at 4 °C for 45 min and centrifugated at 14,000× g for 15 min at 4 °C. The supernatants were incubated with 2 μg primary antibody and shaken slowly at 4 °C overnight. Then, 30 μL protein G agarose (Beyotime Biotechnology, Shanghai, China) was added and shaken slowly at 4 °C for 3 h, followed by centrifugation at 2500 rpm for 5 min. Pellets were washed three times with PBS. After the last centrifugation, 5 × SDS-PAGE Protein Loading Buffer and RIPA buffer were added to resuspend pellets. The mixture was boiled at 95 °C for 8 min. Samples were detected by Western blotting.
8
Co-Immunoprecipitation Assay Protocol
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According to the instruction of Protein G Agarose (P2009) (Beyotime, Shanghai, China), Co-IP assay was carried out as follows. Briefly, the co-transfected cells in 100 mm cell culture dishes were washed once with pre-chilled PBS (pH7.0) buffer, and then lysed using 500 μL of RIPA lysis buffer (P0013B) (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 30 min. After centrifugation, the clarified supernatant was obtained and incubated with 2 μg specific antibodies overnight at 4 °C and then uploaded to 20 μL Protein G Agarose. Next, the mixture was centrifuged and the precipitation was washed with PBS. Finally, the immunoprecipitates were analyzed using western blotting.
9
IgG Purification from Rat Serum
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Total IgG was purified from the serum of SD rats with Protein G Agarose (Beyotime, Jiangsu, China) according to the manufacturer's instructions. The concentrations of IgG were measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
10
Monoclonal Antibody Production Targeting Recombinant Goat CD4 and CD25
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Expression vector PET32a and PGX-6P-1 were preserved in our laboratory. Recombinant expression and affinity column purification of the extracellular region of goat CD4 and CD25 genes were conducted in E. coli (Gao et al. 2019 (link); Yang et al. 2019 (link)). Two recombinant strains were successfully constructed using expression vectors CD4-PGX-6P-1 and CD25-PET32a, which were named as “rCD4” and “rCD25.” Six-week-old female BALB/c mice were immunized four times with purified recombinant proteins, and the procedures for immunization and cell fusion using PEG1500 (Sigma-Aldrich) were performed as previously described (Wagner et al. 2006 (link)). Hybridoma lines producing mAbs that only recognized the recombinant proteins but not the tag protein were screened by ELISA. Positive hybridoma cells that had been cloned three times were used to make ascites (Li et al. 2019b (link); Liu et al. 2022 (link); Ma et al. 2019 (link)). The ascites of mice were purified by protein G agarose (Beyotime, Shanghai, China), labeled with a conjugation kit (Bioss, Beijing, China).
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