The whole hippocampus was dissected and mechanically homogenized, then lysed in appropriate volume of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing phenylmethanesulfonyl fluoride (PMSF) (YEASEN) for 1 h on ice, and cleared by centrifugation for 10 min at 15000 g at 4 °C. Protein concentrations of the lysate were determined using Bradford reagent (Bio-Rad, Hercules, CA, USA).
For co-immunoprecipitation, the lysate was precleared with agarose slurry, and incubated with PRG-1 antibody (1:100, synaptic system), then pulled down by Protein G agarose resin (absin). Finally, beads were suspended with appropriate amount of lysis buffer and analyzed by western blot.
For western blot , tissue lysates or immunoprecipitated samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (Biosharp). Then membranes were incubated with first antibodies, including PRG-1 (1:3000, synaptic system), P2X7R (1:1000, abcam), PPP2R2A (1:1000, Cell Signaling), ß-actin (1:5000, MP Biomedicals), and horseradish peroxidase (HRPO)-conjugated secondary antibodies (1:5000, dianova). Finally, membranes were developed by ECL (EpiZyme scientific).
Protein g agarose resin
Manufactured by Absin
Sourced in United States
Protein G agarose resin is a chromatography medium used for the purification of immunoglobulins. It consists of Protein G, a bacterial cell wall protein, covalently coupled to agarose beads. Protein G has a high affinity for the Fc region of immunoglobulins, allowing for the selective capture and purification of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Biosharp (2 mentions)
Horseradish peroxidase hrpo conjugated secondary antibodies, by Dianova (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Yeasen (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
P2x7r, by Abcam (1 mentions)
Ppp2r2a, by Cell Signaling Technology (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
β actin, by MP Biomedicals (1 mentions)
2 protocols using protein g agarose resin
1
Hippocampal Protein Interactions: Co-immunoprecipitation and Western Blot
2
Co-immunoprecipitation and Western Blot Analysis
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The dissected and mechanically homogenized whole hippocampus or HEK-293 cells, were lysed in appropriate volume of RIPA lysis buffer (Beyotime, China) containing PMSF (YEASEN) for 1 h on ice, and cleared by centrifugation for 10 min at 15,000 g at 4°C. Protein concentrations of the lysate were determined using Bradford reagent (Bio-Rad, Hercules, CA, USA).
For co-immunoprecipitation, the lysate was first cleared with agarose slurry, incubated with PRG-1 antibody (1:100, synaptic system) or normal rabbit IgG (1:100, Millipore) as control, then pulled down by Protein G agarose resin (absin). Finally, beads were suspended with appropriate amount of lysis buffer and analyzed by western blot.
For western blot, tissue or cell lysates were separated by 10% SDS-PAGE and transferred onto PVDF membrane (Biosharp). Membranes were then incubated with first antibodies, including PRG-1 (1:3000, synaptic system), GluR2 (1:3000, Novus), NSF (1:3000, Abcam), β-actin (1:5000, MP Biomedicals), washed, and then incubated with horseradish peroxidase (HRPO)-conjugated secondary antibodies (1:5000, dianova). Finally, membranes were developed by enhanced chemiluminescence procedure (ECL, EpiZyme scientific). Quantification of immunosignals was performed using ImageJ.
For co-immunoprecipitation, the lysate was first cleared with agarose slurry, incubated with PRG-1 antibody (1:100, synaptic system) or normal rabbit IgG (1:100, Millipore) as control, then pulled down by Protein G agarose resin (absin). Finally, beads were suspended with appropriate amount of lysis buffer and analyzed by western blot.
For western blot, tissue or cell lysates were separated by 10% SDS-PAGE and transferred onto PVDF membrane (Biosharp). Membranes were then incubated with first antibodies, including PRG-1 (1:3000, synaptic system), GluR2 (1:3000, Novus), NSF (1:3000, Abcam), β-actin (1:5000, MP Biomedicals), washed, and then incubated with horseradish peroxidase (HRPO)-conjugated secondary antibodies (1:5000, dianova). Finally, membranes were developed by enhanced chemiluminescence procedure (ECL, EpiZyme scientific). Quantification of immunosignals was performed using ImageJ.
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