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7 protocols using rabbit igg 2729

1

Protocatechuic Aldehyde Bioassay Protocol

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Protocatechuic aldehyde (purity ≥ 98%) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). PhosSTOP (4906837001) and EDTA-free, EASYpack protease inhibitor (4693116001) were obtained from Roche (Shanghai, China). Pronase (10165921001) and isoprenaline (I5627) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Trypsin (0.25%), protein A/G magnetic beads (Pierce™, 88802) and Mito-Tracker Red FM (M22425) were all obtained from Thermo Fisher Scientific (Shanghai, China). The H2DCFDA (HY-D0940) fluorescence probe was obtained from MCE (Shanghai, China). PNU-74654 (S8429) was obtained from Selleck (Shanghai, China). Antibodies against PKM2 (4053), SIRT6 (12486), cleaved caspase 3 (9664s), α-actinin (6487), mouse anti-rabbit IgG (light-chain specific, D4W3E) mAb (93702) and the rabbit IgG (2729) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH (60004-1-Ig), β-catenin (51067-2-AP, 66379-1-Ig), PCNA (10205-2-AP), caspase 3 (66470-2-Ig), BAX (60267-1-Ig), BCL-2 (60178-1-Ig), PKM2 (60268-1-Ig), β-tubulin (10068-1-AP) and TCF4 (22337-1-AP) were obtained from Proteintech (Wuhan, China). Annexin V-FITC apoptosis detection Kit was obtained from Beyotime (C1062M, Suzhou, China).
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2

ChIP Assay Protocol for Histone Modifications

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ChIP assay was performed using Zymo-Spin ChIP kit according to the manufacturer’s protocol (Zymo Research, Irvine, CA). Briefly, FLS were serum-starved for 24h with DMEM/0.1%FCS with penicillin, streptomycin, gentamicin and glutamine). The cells were fixed in 1% formaldehyde (ThermoScientific, Waltham, MA) for 8 minutes at RT. After sonication, the chromatin was immunoprecipitated with specific antibodies overnight at 4°C. The antibodies used were anti-H3K4me1 (ab8895, Abcam, Cambridge, MA), anti-RNA polymerase II (ab26721) or rabbit IgG (2729, Cell Signaling Technology, Danvers, MA). The complexes were then immunoprecipitated with protein A magnetic beads for 1h at 4°C, and reverse-crosslinked at 65°C overnight. The eluted DNA was purified and used as template in the qPCR reaction. A 10% input sample was used as control. The enhancer primers (EpiTect ChIP qPCR primer GPH1007377(-)07A) were purchased from Qiagen (Valencia, CA). Control GAPDH promoter primers were purchased from Clontech (Mountain View, CA).
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3

Erlotinib Modulates PKM2/PKM1 Ratio

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Erlotinib was purchased from Selleckchem (OSI-744, Houston, TX, USA). Human recombinant proteins PKM2 (SAE0021), PKM1 (SRP0415), and EGF (E9644); an anti-β-actin (A2228) antibody; PSAT1 shRNA (TRCN0000291729) and control shRNA (SHC202) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PKM2 (4053), PKM1 (7067), Oct-1 (8157), 𝑎-tubulin (3873), DYKDDDDK-Tag (2368) and rabbit IgG (2729) were obtained from Cell Signaling Technology (Danvers, MA, USA). An anti-PSAT1 (10501-1-AP) antibody was purchased from Proteintech Group Inc. (Rosemont, IL, USA). The PSAT1 Double Nickase CRISPR Plasmid system (sc-403001-NIC) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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4

Signaling Mechanisms in Cancer Cells

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Heregulin β1 (HRGβ1) and SDF-1 were purchased from R&D Systems, Inc. (Minneapolis, MN). Gefitinib and paclitaxel were from Selleck Chemicals, Inc. (Houston, TX). Antibodies used for western blot assays were from the following: HER3 (12,708), p-HER3 (Y1286), EGFR (4267), p-EGFR (Y1068), p-Akt (S473), Akt (9272), IGF-1R (3027), PARP (46D11), caspase-8 (1C12), caspase-3 (8G10) (Cell Signaling Technology, Inc., Danvers, MA); β-actin mouse mAb (clone AC-75) (Sigma-Aldrich, Inc., St. Louis, MO). Antibodies used for immunoprecipitation assays were from the following: HER3 (12,708), EGFR (4267), mouse IgG1 isotype control (5415), and rabbit IgG (2729) (Cell Signaling Technology, Inc.). Antibodies used for flow cytometry analysis were mouse anti-HER3 mAb (10,201-MM01) from Sino Biological, Inc. (Beijing, China). APC-labelled goat anti-mouse IgG Ab (405,308) from Biolegend Co. (San Diego, CA). All other reagents were from Sigma-Aldrich, Inc. unless otherwise specified.
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5

Immunohistochemical analysis of human heart

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Human heart samples were fixed in 4% paraformaldehyde overnight followed by conventional dehydration and slicing. Then, the human heart samples sections (5 μm) were blocked with 3% hydrogen peroxide and then performed at 95 °C for 10 min using citrate buffer (Beyotime, Shanghai, China), then blocking steps were carried out using the QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) according to the manufacturer's instructions. After incubated with primary antibody at 4 °C overnight, the sections incubated with secondary antibody (G1210, Servicebio, Wuhan, Hubei, China) at 37 °C for 30 min. Visualization was accomplished using 3,3N-diaminobenzidine tertrahydrochloride (G1211, Servicebio, Wuhan, Hubei, China). Sections were counterstained with hematoxylin (Servicebio, Wuhan, Hubei, China). Rabbit IgG (2729S, Cell signaling technology, Beverly, MA, USA) was used instead of a primary antibody in the negative control group. The mean density was quantified from 4-6 fields per sample with the Image-pro plus software program (Media Cybernetics, Rockville, MD, USA). The antibodies are listed in Table S1.
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6

FoxO1 ChIP-qPCR Analysis of PNPLA2

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Chromatin immunoprecipitation (ChIP) was performed as previously described [24 (link)]. Briefly, chromatin was precipitated with FoxO1 antibody (rabbit mAb C29H4, Cell Signaling Technology) and the relative IgG control (rabbit IgG 2729S, Cell Signaling Technology). 1% of chromatin used for immunoprecipitation was kept as input control. Purified DNA was analyzed by RT-qPCR. Each RT-qPCR value was normalized over the appropriate input control and reported in graphs as a ratio over the IgG. The analyzed amplicon of the PNPLA2 genome mapped on FoxO1 binding site as highlighted by Jaspar TF tool in UCSC genome browser (Fig. 6D). Primers for PNPLA2 region for 5- TTCATGGGTGAGGGTGCTTC − 3 and rev: 5- ACATCACTCCCTCATGGCAG − 3.
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7

ChIP-seq Transcription Factor Protocol

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ChIP was performed following Abcam’s crosslinking ChIP protocol with an iDeal ChIP-seq Kit for Transcription Factors (C01010055; Diagenode). In brief, proteins and chromatin in cells were crosslinked with 0.75% formaldehyde for 20 min. The crosslinked chromatin was sonicated with an ultrasonic processor (Sonics; VCX 130 PB) to shear DNA into fragments of 150–300 bp. Protein A-coated beads were coupled to 2 μg of each antibody: anti-FOXM1 (sc-376471; Santa Cruz Biotechnology), anti-E2F1 (sc-193; Santa Cruz Biotechnology), mouse IgG (sc-2025; Santa Cruz Biotechnology), and rabbit IgG (2729 S; Cell Signaling Technology). The antibody-coupled beads were subsequently incubated with the lysate at 4 °C overnight. Next, the retrieved complexes were washed and eluted in buffer containing 100 mM NaHCO3 and 1% SDS, and DNA was recovered by reverse crosslinking at 65 °C overnight. DNA was then extracted by ethanol precipitation. The primer sequences used for qPCR are listed in Supplementary Data 6.
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