Primary antibodies used were: anti-Actin (Abcam, ab3280), anti-SETD6 (Genetex, GTX629891), Anti-SETD3 (ab176582; Abcam), HRP-conjugated secondary antibodies, goat anti-rabbit, goat anti-mouse, (were purchased from Jackson ImmunoResearch (111-035-144, 115-035-062 respectively). Fluorescently labeled secondary antibodies used: Alexa 647 anti-rabbit and antimouse (Invitrogen, A-21443). For Western blot analysis, cells were homogenized and lysed in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, and 1:100 protease inhibitor mixture (Sigma)). Samples were resolved on SDS-PAGE, followed by Western blot analysis.
Ab176582
Manufactured by Abcam
Ab176582 is a Rabbit polyclonal antibody that recognizes an unspecified target. It is intended for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab176582
1
Western Blot Analysis of Actin, SETD6, SETD3
2
Immunoprecipitation and Western Blot Analysis
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Primary antibodies used were as follows: SETD3 (ab176582; Abcam), FoxM1 (GTX102170; GeneTex, HPA029974; Sigma), FLAG (F1804; Sigma), HA (05-904; Millipore), Actin (ab3280; Abcam), H3 (ab10799; Abcam), and pan-methyl (ab23366; Abcam). Secondary HRP-conjugated antibodies (goat anti-mouse and goat anti-rabbit) were from Jackson ImmunoResearch (115-035-062 and 111-035-144, respectively). Coomassie stain was purchased from Expendon (ISB1L).
Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS (v/v), 1 mM dithiothreitol (DTT) and Sigma protease inhibitor cocktail (P8340, diluted 1:100)). Lysates were incubated for 1 h at 4 °C with 10 μl protein A/G beads (Santa Cruz Biotechnology) as a pre-clear step. Pre-cleared lysates were incubated overnight at 4 °C with FoxM1 antibody (1 μg) or pan-methyl antibody (4 μg) conjugated to beads or beads only as a control. For over-expression experiments, cells were lysed as described above and incubated with FLAG-M2-affinity gel beads (A2220; Sigma). After incubation, beads were washed 4 times with lysis buffer, heated at 95 °C for 5 min in Laemmli sample buffer, and resolved by SDS-PAGE.
Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS (v/v), 1 mM dithiothreitol (DTT) and Sigma protease inhibitor cocktail (P8340, diluted 1:100)). Lysates were incubated for 1 h at 4 °C with 10 μl protein A/G beads (Santa Cruz Biotechnology) as a pre-clear step. Pre-cleared lysates were incubated overnight at 4 °C with FoxM1 antibody (1 μg) or pan-methyl antibody (4 μg) conjugated to beads or beads only as a control. For over-expression experiments, cells were lysed as described above and incubated with FLAG-M2-affinity gel beads (A2220; Sigma). After incubation, beads were washed 4 times with lysis buffer, heated at 95 °C for 5 min in Laemmli sample buffer, and resolved by SDS-PAGE.
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