GFP-positive HeLa cells transfected with the indicated plasmids were collected using FACS (BeckMan Coulter, Astrios EQ). Generally, 106 cells were sorted, washed twice with PBS, and lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% NP-40, 0.1%SDS and protease inhibitors. After incubation at 4 °C for 10 min, the soluble fraction (containing 0.1 M NaCl) was separated from the chromatin fraction by centrifugation at 15,000 × g for 10 min. The chromatin pellet was then incubated in the above buffer with the addition of 0.5 M NaCl at 4 °C for 1 h, followed by centrifugation at 15,000 × g for 10 min. The final pellet was resuspended in 1 M NaCl of the above buffer, sonicated for 20 s (1 s on and 1 s off), incubated at 4 °C for 1 h, and then centrifuged. These lysate fractions were subjected to Co-IP with an antibody against SMC3 (abcam, ab9263). Further immunoblot analyses were performed with the following antibodies: H3 (Ruiying Biological Technology, RLM-3038), ACTB (ABclonal, AC026), RAD21 (Thermo Scientific, PA5-54128), and SMC3 (abcam, ab9263).
Ab9263
Manufactured by Abcam
Sourced in United Kingdom
Ab9263 is a primary antibody that recognizes the human GFAP protein. It is suitable for applications such as Western Blotting and Immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab992, by Abcam (4 mentions)
Ab11174, by Abcam (2 mentions)
Nb110 57130, by Abcam (2 mentions)
A2228 100ul, by BD (2 mentions)
Sc 6216, by Santa Cruz Biotechnology (2 mentions)
Ab1791, by Abcam (2 mentions)
Ab70303, by Abcam (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Na934v, by GE Healthcare (2 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Gradient sds polyacrylamide gel, by Bio-Rad (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo western transfer system, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab171870, by Abcam (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Pharmingen transcription factor buffer set, by BD (1 mentions)
Ab290, by Abcam (1 mentions)
18 protocols using ab9263
1
Chromatin Immunoprecipitation and Immunoblot Analysis
2
Western Blot Analysis of Protein Levels
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For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a BCA protein assay kit (Pierce). Lanes of 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. Following protein separation via standard SDS PAGE, proteins were transferred to PVDF membranes using the Trans-Blot® Turbo™ western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2 . At a 1∶20,000 dilution, Invitrogen horseradish peroxidase-conjugated antibodies rabbit anti-mouse (R21455), goat anti-rabbit (A10533), rabbit anti-goat (R21459) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH). The SMC3 Co-IP experiment was performed using the Dynabead® Co-IP kit (Life Technologies). Each milligram of beads was covalently linked to 4 µg of SMC3 antibody (Abcam, ab9263) or corresponding IgG control antibody (Life Technologies, A10533).
3
Immunofluorescence Analysis of Meiotic Proteins
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The following antibodies were used: rabbit antibodies against TERB1 (Shibuya et al., 2014 (link)), TERB2 (Shibuya et al., 2015 (link)), MAJIN (Shibuya et al., 2015), SYCE3 (Zhang et al., 2019 (link)), TRF2 (Novus Biologicals; NB110–57130), γH2AX (Abcam; ab11174), and SMC3 (Abcam; ab9263); mouse antibodies against TRF1 (Shibuya et al., 2014 (link)), β-actin (Sigma; A2228–100UL), and MLH1 (BD Biosciences; 51–1327GR); rat antibody against RPA2 (Cell Signaling Technology; 2208); guinea pig antiserum against histone H1T (Inselman et al., 2003 (link)); goat antibody against Lamin B (Santa Cruz Biotechnology; sc-6216); and chicken antibody against SYCP3 (Zhang et al., 2019 (link)).
4
Isolation and Analysis of SMC3-bound RNAs
5
Intracellular SMC3 Detection in BM Cells
6
Immunoprecipitation Protocol for GFP and SMC3
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Extracts from 12 × 103 cells were prepared as previously described. Two microlitre of extract were incubated with 180 ng of GFP antibody (HPLC purified Abcam Ab290: Ab6556) or Rabbit Control IgG (Abcam 46540, Lot.GR63822-2, ChIP grade). Extracts from HeLa Kyoto cells were incubated with anti-SMC3 antibody (Abcam Ab9263, ChIP grade) or normal rabbit IgG (Santa Cruz sc-2027) at 90 ng/4 µg antibody/extract ratio. Extract and antibody were incubated for 1 h at 4 °C before proceeding with loading into the microfluidics device.
7
Antibody-Mediated Cohesin Depletion in Meiosis
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The anti-Smc3 antibody used was rabbit anti-Smc3 (Abcam ab9263). The anti-Rec8 antibody was generated in-house using a previously characterized epitope [47 (link)]. The control IgG used was a normal rabbit IgG (Millipore 12-370). With the exception of anti-Smc3, all antibodies were concentrated using Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace the buffer with PBS. Following concentrations of antibodies were used: anti-Smc3 (1 mg/ml), anti-Rec8 (2 mg/ml) and control IgG (2 mg/ml). Prior to microinjection into eggs, the antibodies were spun at 10,000 rpm (4°C) for 10 minutes and supplemented with NP-40 at a final concentration of 0.05%. Antibody microinjection into eggs was performed as described previously for mRNA microinjection [52 (link)]. For full depletion experiments in the metaphase of meiosis II, a bolus of 6 pl of anti-Smc3 or anti-Rec8 was microinjected into the eggs, whereas for partial depletion experiments 2 pl of the anti-Smc3 antibody were microinjected. For partial depletion of cohesins in meiosis I, a bolus of 4 pl of the anti-Smc3 antibody was microinjected 4.5-5.5 hours after the oocytes were released from prophase arrest. The oocytes were then fixed 7 h 15 min – 8 hours after the release.
8
Chromatin-Enriched Protein Extraction Protocol
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Embryos were washed with PBS and lysed on ice for 10 min in a lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM MgCl2, 10% [v/v] glycerol, 0.2% [v/v] NP-40, and 1 mM NaF). Chromatin-enriched fractions were collected by low-speed centrifugation at 1500g for 5 min, washed with the lysis buffer, and further lysed with 1× SDS buffer. Proteins were separated using polyacrylamide gels (5.5% for SMC3 and 12.5% for histone H3) and transferred onto polyvinylidene difluoride membrane (MilliporeSigma). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated with primary antibodies (SMC3: abcam ab9263, histone H3: abcam ab1791) overnight at 4°C. After several washes, membranes were incubated with secondary antibody (anti-rabbit IgG Peroxidase, Sigma-Aldrich A0545) for 1 h at room temperature. Protein bands were visualized using the ECL Select Western Blotting Detection Reagent (GE Healthcare) and detected by ImageQuant (GE Healthcare).
9
Protein Extraction and Western Blot Analysis
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Fully grown GV-stage oocytes from each group were isolated as mentioned earlier, and briefly washed with L-15 medium. Then, 30 oocytes per tube were collected in 10 µl of L-15 medium and snap-frozen in liquid nitrogen. Frozen oocytes were stored at −80°C before testing. Proteins were extracted using Bio-Rad sample buffer (161-0747) with 2-mercaptoethanol (Sigma-Aldrich; M7522) at 95°C for 10 min and then centrifuged at 16,000 g for 2 min; the supernatant was then collected. Proteins were separated using Bio-Rad 4-15% gradient gels (456-1084) and blotted on a PVDF membrane (GE Healthcare; 10061-494). Blots were blocked with 0.3% ECL Prime Blocking Reagent (GE Healthcare; RPN418) in Tris-buffered saline supplemented with 0.01% Tween (TBSTw) for 1 h. SMC3 (Abcam; ab9263; 1:2000) and γ-tubulin (Abcam; ab11316; 1:1000) primary antibodies were diluted in 0.3% blocking buffer at 4°C overnight. Blots were washed with TBSTw three times for 10 min each and then incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (GE Healthcare; NA931V and NA934V, respectively; 1:5000). Blots were washed with TBSTw three times each for 10 min and then developed with a ECL Prime Western Blotting Detection Reagent kit (GE Healthcare; RPN2232).
10
Immunoprecipitation of Nuclear Proteins
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Nuclei were isolated from MEL cells and lysed to make nuclear extracts. Nuclear extracts were then incubated with the indicated antibodies overnight at 4 °C. Protein G/A magnetic beads (Thermo Scientific) equilibrated with IP buffer (20 mM HEPES pH 7.9, 25% glycerol, 200 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.02 % NP-40) were added and the mixture was incubated for an additional 2 h at 4 °C. Beads were washed four times for 15 min and eluted by boiling in 2X Laemmli sample buffer.
The protein expression was detected as described66 . Briefly, cells were lysed in RIPA buffer (Boston Bioproducts) containing 1 mM DTT, 1 mM PMSF, and protease inhibitors and analyzed by SDS-PAGE and Western blotting using specific antibodies. For immunoprecipitation assays, protein extracts were mixed with Laemmli buffer and boiled at 95 °C for 5 min, followed by SDS-PAGE. Following antibodies diluted 1:1000 were used: Esco2 (bethyl, A301-689A), Matr3 (Abcam, ab84422), CTCF (Abcam, ab70303), Rad21 (Abcam, ab992), Smc3 (Abcam, ab9263), Mbd1 (Abcam, ab187734). Uncropped blots are provided in Source data.
The protein expression was detected as described66 . Briefly, cells were lysed in RIPA buffer (Boston Bioproducts) containing 1 mM DTT, 1 mM PMSF, and protease inhibitors and analyzed by SDS-PAGE and Western blotting using specific antibodies. For immunoprecipitation assays, protein extracts were mixed with Laemmli buffer and boiled at 95 °C for 5 min, followed by SDS-PAGE. Following antibodies diluted 1:1000 were used: Esco2 (bethyl, A301-689A), Matr3 (Abcam, ab84422), CTCF (Abcam, ab70303), Rad21 (Abcam, ab992), Smc3 (Abcam, ab9263), Mbd1 (Abcam, ab187734). Uncropped blots are provided in Source data.
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