Western Blots were performed using the following antibodies: WAPL (Santa Cruz, A-7), SCC4 (Abcam, ab46906), SCC2 N-terminal (Santa Cruz, C-9), SCC2 C-terminal (Absea, serum of KT55), HSP90 (Santa Cruz, H-114), CDK4 (Santa Cruz, C-22) and Actin (Santa Cruz, I-19). All antibodies were used at 1:1000 dilution. Secondary antibodies Goat anti-Rabbit-PO, Goat anti-Mouse-PO, Rabbit anti-Goat-PO (DAKO) and Rabbit anti-Rat-PO (Santa Cruz, sc2006) were used at 1:2000 dilution. For immunofluorescence we used the SCC1 (Millipore, 05-908) antibody in a 1:100 dilution. CTCF (Millipore, 07-72), SMC1A (Bethyl, A300-055a) and IgG Rabbit (Sigma, I5006) were used for chromatin immuno-precipitations.
Igg rabbit
Manufactured by Merck Group
The IgG rabbit is a laboratory equipment product used for the detection and quantification of immunoglobulin G (IgG) in samples. It functions as an antibody-based tool for analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Igg mouse, by Merck Group (2 mentions)
Goat anti rabbit po, by Agilent Technologies (2 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse po, by Agilent Technologies (1 mentions)
Smc1a, by Fortis Life Sciences (1 mentions)
Gfp pab, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose beads, by Cytiva (1 mentions)
Protease inhibitor complex, by Roche (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
4 protocols using igg rabbit
1
Antibody Validation for Protein Analysis
2
Immunoprecipitation and Western Blotting
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Cells seeded in flasks were trypsinized, washed with PBS, and lysed using Hunt’s buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40) for 30 min on ice. For IP analysis, 1 mg lysate was continuously mixed with 2 μg antibody overnight at 4°C. Antibodies used were GFP pAb (Invitrogen, Thermo Fisher Scientific), APC Ab5 mAb (Merck Millipore, Darmstadt, Germany), APC C20 pAb (Santa Cruz), Miro1 pAb (Sigma-Aldrich, St. Louis, MO), immunoglobulin G (IgG) mouse (Sigma-Aldrich), and IgG rabbit (Sigma-Aldrich). Lysates were continuously mixed for an additional 2 h at 4°C with 30 μl protein A-Sepharose beads (Amersham Biosciences, GE Healthcare) to pull down immunocomplexes. Immunocomplexes attached to the beads were pelleted by centrifugation and washed three times with TBS. Precipitates were boiled in Laemmli buffer and subjected to SDS–PAGE and Western blotting as described below.
3
Immunoprecipitation of Protein Complexes
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One 15-cm plate containing cells grown to 80% confluence was used for each treatment condition and divided into aliquots for different immunoprecipitation conditions or antibodies. To harvest, cells were washed twice with PBS on the plate, then lysed on ice using a cell scraper with lysis buffer (150 mM KCl, 0.2% (v/v) NP-40, 10% (v/v) glycerol (v/v), 20 mM Tris pH 7.5, 0.5 mM DTT) containing freshly added protease inhibitor complex (Roche). Following 15 min incubation on ice, lysates were cleared at high speed centrifugation at 14,000 rpm for 10 min before protein quantitation using the BCA assay (Pierce). Equal amounts of lysate (500 μg– 1 mg) were immunoprecipitated while rotating at 4°C with 3 μg of one of the following antibodies: Anti-Flag M2 affinity Gel (Sigma), PP4-C and PP4R2 (Bethyl) or IgG controls: IgG-Mouse or IgG-Rabbit (Sigma). Protein G agarose beads (30 μl) were added to each immunoprecipitate and rotated overnight. Beads were washed four times with lysis buffer, and then resuspended in SDS loading dye and boiled prior to immunoblotting.
4
Chromatin Immunoprecipitation of Histone Acetylation
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Ishikawa and Hec50 cells were grown in stripped media for 24 h in T25 flask. SAHA treatments at 2.5 μM were added for 48 h. Following that time, approximatively 5 × 106 cells for each cell line were processed for ChIP analyses following manufacturer’s instructions (Chromatrap® ChIP-seq, Norfolk, UK) [67 ]. The following antibodies were used; Active Motif, AcH3 (#39140, ChIP-seq); Sigma-Aldrich, IgG rabbit (#12-370, ChIP-seq), IgG mouse (#12-371, ChIP). PCR was used as the endpoint assay to analyze the ChIP DNA, using a BioRad CFX real time PCR machine (BioRad, Watford, UK). The primers sequences used were P21 Forward 5′-CCCACAGCAGAGGAGAAAGAA-3′, P21 Reverse 5′-CTGGAAATCTCTGCCAGACA-3′; P53 Forward 5′-AGACCAGCCTGACCAATA-3′, P53 Reverse 5′-GCTCCCGGAAACATCTCAC-3′. The human GAPDH primers present in the Chromatrap® ChIP-seq kit was used as a positive control. The ratios of the signals in immunoprecipitated DNA versus input DNA was calculated as previously shown [68 (link)].
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