ChIP was performed using a SimpleChIP® Enzymatic Chromatin IP Kit (9003S, Cell Signaling Technology). For ChIP-qPCR, HESCs at 30–50% confluence were plated on 15 cm culture dishes and transfected with either control shNC virus or shNSD2 virus (MOI = 20). About 16 h later, the medium was changed to 20 mL normal medium, and cells were collected for ChIP experiments after 48 h (~4 × 106 cells for each reaction). ChIP experiments, including sample preparation, nuclei preparation, chromatin digestion, analysis of chromatin digestion and concentration, chromatin immunoprecipitation, elution of chromatin, reversal of cross-links and qPCR were conducted strictly according to the kit instructions (Martin-Martin et al. 2016 (link)). For H3K36me2 occupied regions, ChIP experiments were performed using anti-histone H3 (di methyl K36) antibodies (2 μg for 25 μg of chromatin, ab176921, Abcam). The DNA products were quantified using qPCR. The ChIP primers were designed using Primer6 software and are listed in Supplementary Table 2. The region of the MCM7 promoter containing the H3K36me2-binding sites were amplified using a SYBR Green Kit (Takara). Statistical analyses were performed according to the instruction manual: Percent input = 2% × 2(C[T] 2% Input sample − C[T] IP sample).
Ab176921
Manufactured by Abcam
Sourced in United States
Ab176921 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab192985, by Abcam (3 mentions)
Ab176920, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Ab176916, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Chip kit, by Abcam (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Ab137429, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Ab191387, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Ab223694, by Abcam (1 mentions)
Dab abc detection ihc kit, by Abcam (1 mentions)
Ab500, by Abcam (1 mentions)
9 protocols using ab176921
1
ChIP-qPCR Analysis of H3K36me2 Occupancy
2
Western Blot Analysis of Histone Modifications
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Whole cells or human endometrial tissues were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) including a protease inhibitor cocktail (ThermoFisher Scientific). About 20 μg protein extract were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight. Next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The membranes were visualized using Chemiluminescent HRP Substrate (Millipore). The immunoreactive protein band density was quantified using Image J software (NIH). Raw data of all western blots are provided in Supplementary Fig. 4.
3
ChIP-qPCR of H3K36me2 and H3K27me3 at WNT10B
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Chromatin immunoprecipitation (ChIP) assays were conducted using the ChIP Kit (ab500, Abcam) and primary antibodies against H3K36me2 (ab176921, 2 µg, Abcam) or H3K27me3 (ab192985, 2 µg, Abcam) following the provided manual. The enriched DNA was detected using qPCR as above described with the primers 5′-GTCTGGCTCCATCCTCATCT-3′ (sense) and 5′-CTCTCTCACACACCCTCTCC-3′ (antisense) for WNT10B promoter.
4
Immunoblotting of Chromatin Modifiers
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Cells were lysed in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Abcam, Cambridge, UK). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Anti-NSD1 (sc-130470; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NSD2 (ab137429; Abcam), anti-NSD3 (ab137430; Abcam), anti-H3K36me2 (ab176921, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602; Abcam) antibodies were used as the primary antibodies. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and visualized by enhanced chemiluminescence (ECL) reagent (Millipore, Billerica, MA, USA).
5
Immunofluorescence analysis of histone modifications in oocytes
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Oocytes were fixed for 1 h in 4% formaldehyde in a phosphate buffered saline (PBS, pH 7.4) at room temperature (RT). After permeabilization with 0.2% Triton X-100 for 20 min, oocytes were incubated with primary antibodies diluted in blocking solution overnight at 4 °C, including Kdm2a (Abcam, ab191387, 1:500), H3K36me1 (Abcam, ab176920, 1:1000), H3K36me2 (Abcam, ab176921, 1:1000), and H3K36me3 (Abcam, ab9050, 1:1000). After three washes with PBS, oocytes were incubated with corresponding secondary antibody at RT for 30 min, and stained with propidium iodide (PI, 1 μg/mL in PBS) for 5 min. Labeled oocytes were mounted on slides and obtained images using a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany) with the same parameters. The ZEN imaging and ImageJ software were used to analyze the images.
6
Endometrial NSD2 and H3K36me2 in Recurrent Implantation Failure
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Proliferative-phase endometrial tissues from 10 patients with RIF and 10 FER controls were collected and washed with PBS immediately. Tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and serially sectioned. Immunostaining was performed according to the instruction of the mouse and rabbit specific HRP/diaminobenzidine (DAB) (ABC) Detection IHC Kit (ab64264, Abcam). The slides were incubated with primary antibody against NSD2 (1:100, ab223694, Abcam), H3K36me2 (1:1000, ab176921, Abcam), minichromosome maintenance complex component 7 (MCM7) (1:500, 11225-1-AP, Proteintech), or Rabbit IgG (1:500, #3900, Cell Signaling Technology) diluted in PBS overnight at 4°C. The primary antibodies used in the study were all commercial antibodies reported by other empirical studies (Zhu et al. 2019 (link), Dai et al. 2020 (link), Ganig et al. 2021 (link), Acke et al. 2022 (link)). For each immunohistochemical run, a slice from each group was randomly selected as the negative control. The negative control was tested by incubation with immunoglobulin G (IgG) from the corresponding species. Next day, they were incubated with secondary antibodies, stained with DAB, and counterstained with hematoxylin. The slides were then dehydrated, cleared, and mounted. The images were captured under a microscope. See Supplementary methods for the immunohistochemical scoring method.
7
ChIP Assay for NSD1, H3K36me2, H3K27me3
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ChIP assay was performed with the ChIP Kit (ab500, Abcam) and antibodies against NSD1 (H00064324-M08, 2 µg, Novus Biologicals), H3K36me2 (ab176921, 2 µg, Abcam), or H3K27me3 (ab192985, 2 µg, Abcam) in accordance with the instructions. The enriched DNA was measured by qPCR as described above using the primers 5’-GTCTGGCTCCATCCTCATCT-3’ (sense) and 5’-CTCTCTCACACACCCTCTCC-3’ (antisense) for WNT10B promoter.
8
ChIP-seq analysis of NRIP1, NSD2, and H3K36me2
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According to the instruction manual of the ChIP kit (Cat. No. 492024, Thermo Fisher Scientific, Rockford, IL, USA), the SCC-9 or CAL27 cells were cross-linked in 1% formaldehyde and ultrasonicated to truncate DNA to 200-500 bp fragments. Thereafter, the lysates were reacted with the antibodies of NRIP1 (customized by Sangon Biotech), NSD2 (1:50, ab75359, Abcam), and H3K36me2 (1:50, ab176921, Abcam) overnight for immunoprecipitation, with normal rabbit IgG (A7016, Beyotime Biotechnology Co., Ltd., Shanghai, China) or normal mouse IgG (A7028, Beyotime) used as the NC antibodies. The DNA in the immunoprecipitated complexes was eluted and purified for qPCR analysis.
9
Quantitative Western Blot Analysis
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Total protein in cells or xenograft tumor tissues (see details below) was extracted using the mixture of cell lysis buffer and protease inhibitor. The protein sample was separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with non-fat milk and then probed with the primary antibodies including NRIP1 (1:500, A9955, ABclonal Technology Co., Ltd., Wuhan, Hubei, China), B-cell lymphoma-2 (Bcl-2; 1:500, A0208, ABclonal), Bcl-2-associated X (Bax; 1:500, A0207, ABclonal), NSD2 (1:500, GTX106306, GeneTex Inc., San Antonio, TX, USA), DGCR8 (1:1,000, ab191875, Abcam Inc., Cambridge, MA, USA), H3K36me2 (1:2,000, ab176921, Abcam), and β-actin (1:500, GTX109639, GeneTex) overnight at 4°C. Thereafter, the membranes were further incubated with HRP-conjugated mouse anti-rabbit IgG (1:2,000, D110065, Sangon Biotech) at room temperature for 1 h. The blot bands were developed by enhanced chemiluminescence reagent. The gray value of the protein bands was evaluated by Image J to analyze expression of target proteins.
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