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Af2006

Manufactured by R&D Systems
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The AF2006 is a laboratory equipment designed for protein purification. It is a high-performance liquid chromatography (HPLC) system that can be used to separate and purify a variety of biomolecules, including proteins, peptides, and nucleic acids. The AF2006 features advanced technology and precise controls to ensure accurate and reproducible results.

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Lab products found in correlation

Mini protean tgx precast gel, by Bio-Rad (1 mentions) Image lab software, by Bio-Rad (1 mentions) Ab32575, by Abcam (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Af3075, by R&D Systems (1 mentions) Ab31419, by Abcam (1 mentions) Mouse anti β actin ac 15, by Merck Group (1 mentions) Horseradish peroxidase conjugated anti rabbit and anti mouse, by GE Healthcare (1 mentions) Mnase, by Thermo Fisher Scientific (1 mentions) Mab004, by R&D Systems (1 mentions) Nuclei isolation buffer, by Abcam (1 mentions) Ab23980, by Abcam (1 mentions)

3 protocols using af2006

1

Western Blot Analysis of Cell Signaling Proteins

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Cell lysate supernatants were collected and protein samples (10 μg/lane) were resolved by electrophoresis on 10% Mini-protean TGX precast gels (Bio-Rad Laboratories, Inc., UK). The proteins were wet transferred to a nitrocellulose membrane. Protein bands were visualized by staining with Ponceau S stain and imaged to quantify total lane protein as previously described (26 (link)). Membranes were then incubated in blocking buffer followed by incubation with either rabbit anti-human smooth muscle alpha actin (Abcam, ab32575, 1:2,500 dilution), goat anti-human Runx2 (R&D systems, AF2006, 1:2,000 dilution), or goat anti-human Sox9 (R&D systems, AF3075, 1:400 dilution) overnight at 4°C. The membrane was then washed and incubated for 1.5 h at room temperature with alkaline phosphatase conjugated anti-rabbit secondary antibody (Sigma, Catalog No. A3937, 1:25000 dilution in 3% marvel in TBST) or anti-goat secondary antibody (Abcam, ab97097, 1:5,000 dilution in 3% marvel in TBST). Immunoreactive bands were visualized by chemiluminescence (Bio-Rad Immun-Star™ AP Substrate Pack #1705012). Protein bands were visualized using the ChemiDoc™ MP Imaging system with Image Lab™ software (Bio-Rad). Proteins were normalized to total lane protein as determined by Ponceau S staining.
Millar S.A., John S.G., McIntyre C.W., Ralevic V., Anderson S.I, & O'Sullivan S.E. (2020). An Investigation Into the Role of Osteocalcin in Human Arterial Smooth Muscle Cell Calcification. Frontiers in Endocrinology, 11, 369.
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2

Western Blot Analysis of Transcription Factors

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Western blot analysis was performed as previously described (29 (link)). Antibodies used were rabbit anti-c-JUN (ab31419; Abcam), goat anti-RUNX2 (AF2006, R&D Systems), rabbit anti-BRD4 (A301-985A50, Bethyl), mouse anti-beta-actin (AC-15; Sigma-Aldrich), mouse anti-alpha-tubulin (sc-8035, Santa Cruz), horseradish peroxidase-conjugated anti-rabbit and anti-mouse (GE Healthcare).
Sancisi V., Manzotti G., Gugnoni M., Rossi T., Gandolfi G., Gobbi G., Torricelli F., Catellani F., Faria do Valle I., Remondini D., Castellani G., Ragazzi M., Piana S, & Ciarrocchi A. (2017). RUNX2 expression in thyroid and breast cancer requires the cooperation of three non-redundant enhancers under the control of BRD4 and c-JUN. Nucleic Acids Research, 45(19), 11249-11267.
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3

Chromatin Immunoprecipitation (ChIP) Assay

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ChIP assays were performed as previously described (46 (link)). Briefly, aliquots of 2.5×106 formaldehyde-fixed cells were resuspended in 50 μl nuclei isolation buffer (Abcam, Cambridge, United Kingdom), and chromatin was digested with 15 U MNase (ThermoFisher) for 5 min. at 37ºC. EDTA was added to stop the reaction. After enzymatic digestion, antibodies against Runx1 (ab23980, Abcam), Runx2 (AF2006, R&D Systems, Minneapolis, MN), Runx3 (353604, BioLegend), TET2 (PA5–35847, ThermoFisher), and TET3 (PA5–34431, ThermoFisher) were added to precipitate the sheared chromatin. An IgG isotype antibody (MAB004, R&D Systems) was also added for a background control. After washing steps, protein and DNA complexes were eluted and cross-links were reversed. Purified DNA samples were analyzed by quantitative real time PCR with primers listed in Supp. Table 1.
Wu C.Y., Zhang B., Kim H., Anderson S.K., Miller J.S, & Cichocki F. (2020). Ascorbic Acid Promotes KIR Demethylation During Early NK Cell Differentiation. Journal of immunology (Baltimore, Md. : 1950), 205(6), 1513-1523.
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