Western blot analysis was performed as described previously42 (link). Samples were collected directly in 1 × NuPAGE LDS sample buffer with 1 × Sample reducing buffer (Invitrogen) and denatured at 95 °C for 5 min followed by a centrifugation at 18,500g for 5 min. The supernatant was electrophoresed on a 4–12% Tris-HCl gel and transferred to nitrocellulose membrane (Invitrogen). After blocking with Superblock T20 blocking buffer (Thermo Scientific), the membrane was incubated with a primary antibody overnight at 4 °C and then with a secondary antibody conjugated with alkaline phosphatase (1 h at room temperature); the signal was detected by using a chemiluminescence method. The following primary antibodies were used: anti-SETDB1 (Santa Cruz, 1:1,000), anti-p53 (Cell Signaling, 1:1,000), anti-p53/K370me1 and anti-p53/K370me2 (in-house made as previously described32 (link); 1:1,000), anti-p53/K372me1 (ab16033, AbCAM, 1:1,000), anti-GAPDH (7B; Santa Cruz, 1:10,000), anti-H3K9me3 (39161, Active Motif, 1:1,000), anti-p-p53(S15; 16G8; Cell Signaling, 1:1,000) and anti-MDM2 (1F2; Millipore, 1:200).
Anti setdb1
Manufactured by Santa Cruz Biotechnology
Anti-SETDB1 is a laboratory product that targets the SETDB1 protein. SETDB1 is a histone methyltransferase that catalyzes the tri-methylation of histone H3 at lysine 9 (H3K9me3), a modification associated with transcriptional repression. This product can be used to detect and quantify SETDB1 levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Reducing sample buffer, by Thermo Fisher Scientific (2 mentions)
Superblock t20 blocking buffer, by Thermo Fisher Scientific (2 mentions)
Ab16033, by Abcam (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti gapdh 7 b, by Santa Cruz Biotechnology (1 mentions)
Anti h3k27me2, by Cell Signaling Technology (1 mentions)
Anti suz12, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti histone 3, by Cell Signaling Technology (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Anti h3k9me3, by Active Motif (1 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
2 protocols using anti setdb1
1
Western Blot Analysis of Protein Modifications
2
Western Blot Analysis of Chromatin Regulators
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Samples were collected directly in 1× NuPAGE LDS sample buffer with 1× sample reducing buffer (Invitrogen) and denatured for 5 min at 95°C before being centrifuged for 5 min at 13,200 rpm. Supernatants were electrophoresed on a 4%–12% Tris-HCl gel and transferred to nitrocellulose membrane (Invitrogen). After blocking with superblock T20 blocking buffer (Thermo Scientific), the membrane was incubated with primary antibody overnight at 4°C and, subsequently, with HRP-conjugated secondary antibody (1 h at room temperature). The signal was detected using ECL (Pierce). The following primary antibodies were used: anti-SETDB1 (Santa Cruz, no. 66884; 1:1000), anti-Histone 3 (Cell Signaling, no. 9715; 1:1000), anti-H3K9me3 (Active Motif, no. 39161; 1:1000), anti-H3K27me2 (Cell Signaling, no. 9728; 1:1000), anti-H3K27me3 (Millipore, 07-449; 1:1000), anti-EZH2 (Cell Signaling, no. 3147; 1:1000), anti-SUZ12 (Cell Signaling, no. 3737; 1:1000), and anti-GAPDH (Santa Cruz, no. 137179; 1:10000).
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