Immunoprecipitation of extracts from 293 cells (lane 1 and 3) or 293T cells (lane 2 and 4) using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (lane 1 and 2) or SV40 Large T Antigen (D1E9E) Rabbit mAb (Sepharose® Bead Conjugate) (lane 3 and 4). Lanes 5 and 6 are 10% input. Western blot analysis was performed using SV40 Large T Antigen (D1E9E) Rabbit mAb #15729 as the primary antibody and was visualized using Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127.Learn more about how we get our images.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibody.
SV40 Large T Antigen (D1E9E) Rabbit mAb (Sepharose® Bead Conjugate) recognizes transgenic levels of total SV40 Large T Antigen protein.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys315 of SV40 Large T Antigen protein.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads.
SV40 Large T Antigen (D1E9E) Rabbit mAb (Sepharose® Bead Conjugate) is useful for immunoprecipitation assays. The antibody is expected to exhibit the same cross-species reactivity as the unconjugated SV40 Large T Antigen (D1E9E) Rabbit mAb #15729.
Simian Virus 40 (SV40) is a small double-stranded, circular DNA tumor virus encoding three tumor antigens: large T, Small T, and 17KT. These antigens contribute to cellular transformation, an area of research where they are utilized extensively. Transformation of cells by SV40 large T antigen is mediated by its binding to the tumor suppressor proteins RB and p53 in the nucleus. Additional binding partners include p300, CBP, Cul7, IRS1, Bub1, Nbs1, and Fbw7, which also contribute to transformation (1).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Sepharose is a registered trademark of GE Healthcare.
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|45159S||400 µl (40 immunoprecipitations)||$ 299.0|