Immunoprecipitation assays were conducted as previously described [30 (link),31 (link),32 (link),33 (link)], with some modifications. Briefly, 500 μL of whole-cell lysates collected in RIPA buffer containing a protease inhibitor cocktail was incubated with 50 μL of protein A beads (nProtein A Sepharose 4 Fast Flow; Cytiva, Tokyo, Japan) on a rotating wheel at 4 °C for 1 h to eliminate non-specific interactions. The pre-cleaned lysates were incubated with an anti-PD-L1 Ab or control IgG Ab on a rotating wheel at 4 °C overnight, and then mixed with 50 μL of protein A beads on a rotating wheel at 4 °C for 3 h. Next, the protein A beads were collected and rinsed three times with RIPA buffer, followed by heating at 97 °C for 5 min in 2× sample buffer (NacalaiTesque, Kyoto, Japan) before immunoblotting.
Nprotein a sepharose 4 fast flow
Manufactured by Cytiva
Sourced in Japan
NProtein A Sepharose 4 Fast Flow is a chromatography resin used for the purification of antibodies and Fc-containing proteins. It is composed of cross-linked agarose beads with covalently coupled recombinant Protein A.
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Lab products found in correlation
5 protocols using nprotein a sepharose 4 fast flow
1
PD-L1 Immunoprecipitation Assay Protocol
2
Immunoprecipitation Assay for PD-L1 Analysis
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Immunoprecipitation assays were conducted as previously described [27 (link),28 (link),29 (link),30 (link)], with some modifications. Briefly, 500 μL of whole-cell lysates collected in RIPA buffer containing a protease inhibitor cocktail was filtered through 50 μL of protein A beads (nProtein A Sepharose 4 Fast Flow; Cytiva, Tokyo, Japan) for 1 h at 4 °C on a rotating wheel to eliminate non-specific interactions. The pre-cleaned lysates were incubated on a rotating wheel at 4 °C overnight with an anti-PD-L1 Ab or control IgG Ab (1:30) and mixed with 50 μL of protein A beads on a rotating wheel at 4 °C for 3 h. After incubation, protein A beads were collected and rinsed three times with RIPA buffer, followed by heating at 97 °C for 5 min in 2× sample buffer (Nacalai Tesque) before immunoblotting.
3
Immunoprecipitation of p53 Protein
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Cell lysates were incubated with 2 μg of anti-p53 antibody (DO-1) overnight at 4°C; then, they were mixed with 50 μL of nProtein A Sepharose 4 Fast Flow (Cytiva, catalog no. 17528001) for 3 hours at 4°C and washed with lysis buffer three times. The immunoprecipitated proteins were eluted from the nProtein A-Sepharose beads by boiling with 2× sample buffer (Invitrogen, catalog no. NP0007) and subjected to SDS-PAGE.
4
Co-Immunoprecipitation of Edwardsiella tarda Outer Membrane Proteins
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Co-IP was carried out as described previously (Pan et al., 2011 (link)). Mouse antisera against ETAE_p037 or ETAE_p044 were purified with 33% saturated ammonium sulfate and incubated with a mixture of recombinant ETAE_p037 or ETAE_p044 with E. tarda outer membrane proteins for 12 h at 4°C on a gentle shaker. Then, nProtein A Sepharose 4 Fast Flow (Amersham Biosciences Corp., USA) was added and the solution was incubated for 12 h at 4°C on a gentle shaker. The nProtein A Sepharose 4 Fast Flow was collected by centrifugation at 2000 g for 3 min, cleaned with pH 7.0 Tris-HCl buffer six times for 10 min, incubated in pH 2.4 glycine-HCl buffer for 2 h, adjusted to pH 7.4 with 0.1 M Tris and then subjected to SDS-PAGE. The distinct bands on the gels were analyzed using MALDI/TOF-TOF analysis. OmpF2 and OmpA were further identified by Western blot using mouse anti-OmpF2 and anti-OmpA as the primary antibodies and anti-mouse-IgG-HRP as the secondary antibody as described above.
5
Co-Immunoprecipitation of Cathepsin K and E. piscicida Proteins
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Co-IP was carried out as described previously53 . Recombinant cathepsin K and E. piscicida outer membrane proteins were incubated at room temperature for 2 h on a gentle shaker. Then, 10 μL of mouse antiserum against cathepsin K was added to the test group, and pre-immune antiserum was added to the control. After incubation under the same conditions, 20 μL of nProtein A Sepharose 4 Fast Flow (Amersham Biosciences Corp.) was added, followed by incubation for 12 h at 4 °C on a gentle shaker. The nProtein A Sepharose 4 Fast Flow was collected via centrifugation at 3000 g for 5 min and cleaned six times for 10 min each with pH 7.0 Tris-HCl buffer, followed by incubation in 50 μL of 1 mM pH 2.4 glycine-HCl buffer for 2 h at room temperature. After centrifugation at 3,000 g for 5 min, the supernatant was employed for Western blotting using anti-EvpB, -ETAE_3048 and –ETAE_0245 as the primary antibodies.
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