The nuclear protein from human cardiomyocytes was extracted by use of a nuclear protein extraction kit (Millipore, MA, USA). Monoclonal antibodies were added to 300 μg of isolated nuclear protein and incubated in 4°C overnight. Then, 50 μL of protein A/G-Sepharose bead suspension (Santa Cruz, CA) was added to each sample and gently mixed overnight. The samples were then centrifuged at 12,000 ×g for 30 s; thereafter, the beads were washed three times with 1 mL of IP buffer (Millipore, MA). The recovered beads were resuspended in SDS-PAGE loading buffer, and the supernatants were used for electrophoretic separation and immunoblotting.
Protein a g sepharose bead suspension
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Protein A/G-Sepharose bead suspension is a solid-phase affinity matrix used for the purification of immunoglobulins and other proteins that bind to protein A or protein G. The beads are composed of cross-linked agarose and covalently coupled with protein A and protein G, which are bacterial cell wall proteins that exhibit high affinity for the Fc region of immunoglobulins.
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Lab products found in correlation
2 protocols using protein a g sepharose bead suspension
1
Nuclear Protein Immunoprecipitation from Cardiomyocytes
2
Immunoprecipitation of 4-HNE-Akt Adduct
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Immunoprecipitation was performed to examine the levels of 4-HNE-Akt adduct. Briefly, protein extracts (1 mg/mL) were incubated with 5 μl primary Akt antibody at 4 °C overnight with gentle agitation. After that, 20 μl of protein A/G-Sepharose bead suspension (Santa Cruz, CA, USA) was added and gently mixed for 1 h at 4 °C. Samples were centrifuged at 1000×g for 30 s, and the pellet was washed in RIPA buffer for 4 times. Finally, the pellet was suspended in 40 μl 2×SDS-PAGE buffer, heated to 100 °C for 5 min, and was used for western blotting analysis.
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