Cells were lysed with modified RIPA buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1% Triton-X, 0.5% Sodium deoxycholate, and 0.1% SDS) supplemented with complete proteinase inhibitor (Roche); lysates were subjected to sonication and clarified by centrifugation. Protein concentrations were determined by Qubit fluorometer (Invitrogen). Protein extracts were separated on 8-10% SDS-PAGE gels, transferred to PVDF membranes, blocked with 5% milk in Tris-buffered saline (TBS: 20 mM Tris–HCl pH 7.5, 100 mM NaCl) or with Intercept (TBS) blocking buffer (LI-COR) and probed overnight at 4 °C with primary antibodies diluted in TBST (TBS plus 0.1% Tween-20) containing 1% milk or in Intercept (TBS) blocking buffer with 0.1% Tween-20: 1:500 rabbit anti-SETDB1 (PtoteinTech 11231-1-AP), 1:500 rabbit anti-PCGF6 (ProteinTech 24103-1-AP), 1:500 rabbit anti-RING1B (Cell Signaling 5694) and 1:2500–5000 mouse anti-ACTB (Invitrogen MA5-15739). Blots were subsequently washed in TBS-T, incubated in IRDye-conjugated secondary antibodies diluted 1:15,000–20,000 in 1% milk in TBS-T or in Intercept (TBS) blocking buffer with 0.1% Tween-20 and 0.01% SDS, washed again in TBS-T and scanned on the ChemiDoc infrared imaging system (BioRad).
Rabbit anti ring1b
Manufactured by Cell Signaling Technology
Rabbit anti-RING1B is a primary antibody that recognizes the RING1B protein. RING1B is a component of the Polycomb repressive complex 1 (PRC1) and plays a role in gene silencing. This antibody can be used to detect and study the RING1B protein in various experimental applications.
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Lab products found in correlation
Complete proteinase inhibitor, by Roche (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Chemidoc infrared imaging system, by Bio-Rad (1 mentions)
Alexa fluor 647 goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti h2ak119ub, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab80892, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti 5mc, by Active Motif (1 mentions)
Rabbit anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti tet1, by Merck Group (1 mentions)
Mouse anti tet1, by Active Motif (1 mentions)
Mouse anti γh2ax, by Merck Group (1 mentions)
Rabbit anti ezh2, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab32423, by Abcam (1 mentions)
Ab12073, by Abcam (1 mentions)
Ab21873, by Abcam (1 mentions)
3 protocols using rabbit anti ring1b
1
Immunoblotting for Chromatin Modifiers
2
Epigenetic Profiling of Pluripotent Stem Cells
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Primary antibodies used in this study include rabbit anti-5hmC (Active Motif, #39769), mouse anti-5mC (Active Motif, #39649), rabbit anti-NANOG (Abcam, ab80892), rabbit anti-H3K9me2 (Abcam, ab1220), rabbit anti-H3K9me3 (Abcam, ab8898), mouse anti-H3K27me3 (Wako, MABI0323), rabbit anti-H2AK119ub (Cell signaling, #8240), mouse anti-γH2AX (Sigma-Aldrich, 07–164), mouse anti-SCP3 (Santa Cruz, sc-74569), rabbit anti-SCP3 (Novus, NB-300-232), mouse anti-HP1γ (Invitrogen, MA3-054), human anti-Centromere Protein (CREST) (Antibodies Incorporated, 15–235), rabbit anti-TET1 (Millipore, 09–872), mouse anti-TET1 (Active Motif, 91172), rabbit anti-EZH2 (Cell signaling, #5246), rabbit anti-GFP antibody (Invitrogen, A11122), and rabbit anti-RING1B (Cell signaling, #5694). The secondary antibodies used in this study included Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG, and Alexa Fluor 647 goat anti-human IgG (Invitrogen).
3
Antibody Characterization for Cell Research
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The primary antibodies used included mouse anti-Nup153, ascites fluid (gift from B. Burke), rabbit anti-Nup153 (raised against hNup153-GST, amino acids 1300–1399), rabbit anti-Parp (9542, Cell Signaling), mouse anti-Oct-3/4 (sc-5279, Santa Cruz Biotechnology), goat anti-Oct-3/4 (sc-8628, Santa Cruz Biotechnology), mouse anti-α-tubulin (T5168, Sigma-Aldrich), mouse anti-Flag (F1804, Sigma-Aldrich), rabbit anti-Nup107 (raised against mNup107-His, amino acids 600–926), rabbit anti-Tuj1 (neuron-specific class III β-Tubulin; PRB-435P, BioLegend), mouse anti-Gapdh (GTX627408, Genetex), rabbit anti-Blbp (Fabp7; ab32423, Abcam), rat anti-Nestin (556309, BD pharmigen), mouse anti-Gata4 (sc-25310, Santa Cruz Biotechnology), rabbit anti-GFAP (Z0334, Dako), goat anti-Foxa2 (AF2400, R&D Systems), mouse anti-α-Sma (A5228, Sigma), rabbit anti-Nup98 (2292, Cell Signaling), rabbit anti-Sox2 (2748, Cell Signaling), mouse anti-V5 (46-0705, Life Technologies), mouse anti-Ring1b (39663, Active Motif), rabbit anti-Ring1b (5694S, Cell Signaling), rabbit anti-Rybp (ab5976, Abcam), rabbit anti-Cbx7 (ab21873, Abcam), rabbit anti-Nup50 (ab151567, Abcam), rabbit anti-Suz12 (ab12073, Abcam), rabbit anti-Lmnb2 (ARP46356; Aviva Systems Biology), rabbit anti-Mel-18 (sc-10744, Santa Cruz Biotechnology), and rabbit anti-Foxo1 (2880S, Cell Signaling).
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