After treatments, NPC cells were washed with PBS and then lysed with cell lysis buffer (CST, USA). Whole cell lysates were centrifuged at 14,000g for 5 min at 4 °C to remove cell debris, and the supernatants were subjected to SDS-PAGE and immunoblotting, as previously described [25 ]. The following antibodies used in this study were purchased from Abcam (USA): rabbit anti-TET1 (ab191698), rabbit anti-TET2 (ab94580), rabbit anti-TET3 (ab139311), mouse anti-GAPDH (ab8245), rabbit anti-flag (ab1162), rabbit anti-PKM antibody (ab131021), and rabbit anti-Histone H3 antibody (ab176842).
Ab94580
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab94580 is an antibody purification column designed to facilitate the efficient separation and purification of antibodies from complex biological samples. The product provides a reliable and reproducible method for antibody purification.
Automatically generated - may contain errors
Lab products found in correlation
Ab191698, by Abcam (5 mentions)
Ab139311, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab109228, by Abcam (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ab1162, by Abcam (1 mentions)
Ab131021, by Abcam (1 mentions)
Ab176842, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Hrp conjugated gapdh monoclonal antibody, by Proteintech (1 mentions)
Pab 053 050, by Diagenode (1 mentions)
Ab48871, by Abcam (1 mentions)
Ab24577, by Abcam (1 mentions)
Ab65258, by Abcam (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Cd81 sc 9158, by Santa Cruz Biotechnology (1 mentions)
Ab10911, by Abcam (1 mentions)
Loading buffer, by Applygen (1 mentions)
Ab8227, by Abcam (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
22 protocols using ab94580
1
Immunoblotting Analysis of TET Proteins
2
Analyzing TET Protein Levels in Immune Cells
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Western blot analysis was performed to analyze the protein levels of TET1, TET2, and TET3 Cells in PBMCs and moDCs, and verify the transfection efficiency of TET1-shRNA. TET1 (1:1,000, ab191698, Abcam, Cambridge, MA, USA), TET2 (1:1,000, ab94580, Abcam), TET3 (1:1000, ab139311, Abcam), and HRP-conjugated GAPDH monoclonal antibody (1:10,000, proteintech) were used in this experiment. The detailed description is presented in the Supplementary Materials .
3
Western Blot for TET2 and HDAC1
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Standard procedures were used for Western blotting31 (link). Primary antibodies used in these experiments were directed against TET2 (ab94580; Abcam) or HDAC1 (Diagenode; pAb-053-050).
4
Comprehensive Antibody Characterization Panel
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The antibodies to Tet1 (ab191698), Tet2 (ab94580), P2rX7 (ab48871), CD146 (ab24577), PDGFRα (ab65258), and OCN (ab10911) were purchased from Abcam (Cambridge, MA, USA). Antibodies to ALP (sc-28904), CD9 (sc-9148), and CD81 (sc-9158) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibody to Runx2 (8486) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody to Tet3 (20602) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-CD34-PE (551387) and SCA1-PE (553108) antibodies were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD105-PE (12-1051-82), CD45-PE (25-0454-82), CD73-PE (12-0739-42), and CD90-PE (15-0902-82) antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Quantifying Tet2 Expression in Muscle Differentiation
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To analyze the amount of Tet2 in cells, C2C12 cells at 0 and 6 d after differentiation induction were washed with PBS and lysed with lysis buffer (Beyotime). After being mixed with loading buffer (Applygen) and boiled for 10 min, denatured protein samples were separated by 8% SDS-PAGE gel and electrotransferred onto PVDF membranes. The membranes were blocked overnight at 4 °C in 5% milk in TBST buffer and then incubated for 2 h at room temperature with primary antibodies against TET2-specific (Abcam, ab94580; 1:500 dilution), myosin heavy chain (DSHB, MF20; 1:2000 dilution) or β-actin (Abcam, ab8227; 1:4000 dilution), followed by incubation with HRP-conjugated anti-rabbit IgG (Applygen; 1:3000 dilution) or anti-mouse IgG (Applygen; 1:4000 dilution) secondary antibodies for 2 h at room temperature. Immune complex were detected using Super ECL Kit (Applygen).
6
ChIP-seq for Transcription Factors in Human Liver
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The SimpleChIP Plus Enzyme Chromatin IP Kit (#9005, CST) was used for chromatin immunoprecipitation (ChIP) assays. We incubated for 20 min at room temperature for cross‐linking of the liver tissue mixture, made from mix of finely minced human liver tissue (10 mg) and 42.5 μL of 37% formaldehyde. We homogenise suspended tissue, washed twice with ice‐cold phosphate‐buffered saline (PBS) using a B‐type Dounce homogeniser, suspended by centrifugation after adding glycine to stop the cross‐linking reaction. Then, resuspended in Kit Buffer A and incubated with micrococcal nuclease for 20 min at 37°C. We disrupted nuclei by sonication, removed debris by centrifugation and treated clarified nuclear extracts with TET2 antibody (ab94580, Abcam, 1:50) or YY1 antibody (ab109228, Abcam, 1:100). Immunoprecipitation with protein G magnetic beads was performed after incubation at 4°C overnight. ChIP‐enriched DNA was analysed by qPCR using specific primers, as described in Table S3 .
7
Protein Expression Analysis in the Nucleus Accumbens
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Immediately after the behavior test, the brains of subjects were removed after euthanasia, and NAc tissues were taken within 1‐mm diameter of the coordinate (+1.41 mm A/P, +0.75 mm M/L, ±4.70 mm D/V from Bregma) point were collected. We homogenized samples in RIPA buffer (Beyotime Biotechnology) containing 1 mM protease inhibitor PMSF (Beyotime Biotechnology). Proteins were resolved in 8% acrylamide sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon‐P; Millipore). Blots were incubated overnight at 4°C in a mixed solution with rabbit polyclonal anti‐TET3 (1:1000; Catalog# ab139311; Abcam), anti‐TET1 (1:1000; Catalog# ab191698; Abcam), anti‐TET2 (1:1000; Catalog# ab94580; Abcam), anti‐TDG (1:1000; Catalog# ab154192; Abcam), and other antibodies (see Table S1 ). Bands were amplified with horseradish peroxidase‐conjugated secondary antibodies (1:2000; Catalog# 7074s; Cell Signaling Technology). The visualization of the membrane was achieved by X‐ray film exposure (ECL kit; Thermo Fisher Scientific). The target protein immunoreactivity was normalized to GAPDH (1:1000; Catalog# 2118s; Cell Signaling Technology) or β‐tublin (1:1000; Catalog# 2146s; Cell Signaling).
8
Flow Cytometric Analysis of Cardiac-Differentiated hESCs
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The cardiac-differentiated hESCs were isolated and analyzed by flow cytometry. Approximately 1 × 105 cells per sample were fixed with 4% paraformaldehyde (Biosharp, China) and washed with staining buffer (1% fetal bovine serum in PBS) (Beyotime, China). The cells were then permeabilized with 100% cold methanol and incubated with primary antibodies, followed by the corresponding secondary antibody. The primary antibodies contained rabbit anti-TET2 (diluted 1:500, Abcam, ab94580) and rabbit anti-cTnT (diluted 1:500, Abcam, ab209813). The secondary antibodies used were Anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (diluted 1:1000, Cell Signaling Technology, #4412) and Anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (Alexa Fluor™ 647) (diluted 1:1000, Invitrogen, A-21244). As shown in Additional file 1 : Fig. S6, cell gating was performed by using isotype controls, such as Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) (diluted 1:1000, Cell Signaling Technology, #2985) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) (diluted 1:1000, Cell Signaling Technology, #2975). The samples were acquired using Amnis Image StreamX MarkII flow cytometer (Merck & Millipore, Germany) and analyzed with IDEAs.6.2 software.
9
Chromatin Immunoprecipitation for Tet2 and Dnmt1
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Nuclear lysate was sonicated to make small DNA fragments ranging from 100–500 base pairs and then incubated with anti-Tet2 (ab94580, Abcam, UK) or anti-Dnmt1 Ab (H-300, Santa Cruz Biotechnology) overnight at 4°C. Isotype-matched control Ab was used for the negative control. Immune complexes containing DNA fragments were precipitated using Dynabeads (Invitrogen, USA) or EZ-ChIP kit (Milipore, Germany). Relative enrichment of the target regions in the precipitated DNA fragments was analyzed by qPCR. The sequences of primers are as follows.
10
DNA Methylation Regulator Expression
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BEFs were rinsed with PBS and lysed in 150 μL of ice-cold Radio Immunoprecipitation Assay buffer. Proteins were extracted from tissues with cell lysis buffer, boiled for 5 min, and stored at −80°C. Samples were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrically transferred to polyvinylidene fluoride membranes blocked with 5% nonfat dry milk in TBS-Tween 20 (blocking buffer) for 1 h. The membrane was subsequently incubated with the following antibodies, all obtained from Santa Cruz Biotechnology, United States unless otherwise stated: anti-DNMT1 (1:500; sc-271729), anti-DNMT3a (1:500; sc-373905), anti-DNMT3b (1:500; sc-393279), anti-TET1 (1:500; sc-293186) and anti-TET2 (1:1,000; ab94580; Abcam, United States) and anti-TET3 (1:1,000; ab139311; Abcam, United States) or with a polyclonal antibody against α-Tubulin (11224-AP, proteinch, China) at 1:1,000 in TBST at 4°C overnight. Membranes were then washed three times and incubated with a horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibody at 1:5,000 in blocking buffer.
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