Am1010a

Manufactured by Abcepta

The AM1010a is a laboratory instrument designed for the analysis and measurement of various samples. It features high-precision sensors and advanced data processing capabilities to provide accurate and reliable results.

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Lab products found in correlation

Anti sirt1 antibody, by Cell Signaling Technology (1 mentions) Hnrnp l, by Abcam (1 mentions) Trap150, by Fortis Life Sciences (1 mentions) Flag tag (flag), by Cell Signaling Technology (1 mentions) Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)

7 protocols using am1010a

ECDs Glycosylation Analysis in Sf9 Cells

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To investigate whether the α1, α2, β2, and γ2 ECDs were produced as glycosylated proteins in Sf9 cells, a glycoprotein deglycosylation kit (catalog no. 362280, Millipore) was used. Briefly, 20 μg of each ECD was dissolved in 30 μl of deionized water in a microcentrifuge tube along with 10 μl of 5× reaction buffer (250 mm sodium phosphate buffer, pH 7.0) and 2.5 μl of denaturation solution (containing 2% SDS, 1 m β-mercaptoethanol, pH 7.0). Samples were boiled at 95 °C for 5 min and left to cool at room temperature. Then, 1 μl of the following enzymes endo-α-N-acetylgalactosaminidase, α2–3,6,8,9-neuraminidase, β1,4-galactosidase, β-N-acetylglucosaminidase, and N-glycosidase F were added to each of the samples, along with 2.5 μl of Triton X-100 detergent solution. Samples were incubated for 3 h at 37 °C before being analyzed using SDS-PAGE, alongside non-deglycosylated ECDs, as a positive control. Immunoblotting was carried out with a His₆ tag primary antibody (1:1000; catalog no. AM1010a, Abgent) followed by the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2500; catalog no. 31340, Invitrogen). Immunoreactivity was detected using the 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (catalog no. S3771) and nitro blue tetrazolium chloride (catalog no. S3771) (both from Promega; Southampton, UK).

Brown L.E., Nicholson M.W., Arama J.E., Mercer A., Thomson A.M, & Jovanovic J.N. (2016). γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains. The Journal of Biological Chemistry, 291(27), 13926-13942.

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Immunoblot Detection of Recombinant Sirtuins

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Recombinant sirtuins were labeled as described in section 2.3 and resolved by SDS-PAGE, transferred to a nitrocellulose membrane, blocked with 5% w/v milk in PBST, and probed with an anti-His₆ antibody (1:1000 dilution, Abgent AM1010a, lot SG100323A) at 4 °C overnight. The membrane was washed (3 × 5 min, PBST), incubated with secondary antibody for 1 h at ambient temperature, and washed again (3 × 5 min, PBST) before being developed by ECL and imaged on a ChemiDoc MP Imaging System. HEK293T whole-cell lysates and MCF7 subcellular fractions (50 μg) were generated as described in section 2.5 and processed by the same immunoblot protocol. In HEK293T lysates, Sirt1 was detected with anti-Sirt1 antibody (1:1000 dilution, Cell Signaling Technology, 1F3-CST8469). For MCF7 cells, anti-H3 antibody (1:5000 dilution, Abcam 1791, lot GR3236360–1) detected subcellular fractions. Secondary antibodies used were goat anti-rabbit (1:5000 dilution, Thermo A10549, lot UH2818652) or goat anti-mouse (1:5000 dilution, Invitrogen 31437, lot UG2814213). All antibodies were diluted with 5% w/v milk in PBST.

Goetz C.J., Sprague D.J, & Smith B.C. (2020). Development of activity-based probes for the protein deacylase Sirt1. Bioorganic chemistry, 104, 104232.

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Immunoprecipitation and Western Blot Analyses

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The following antibodies were used throughout as noted: PSF (Sigma P2860 for IP, Abnova H00006421-A01 for WB), TRAP150 (A300–956A, Bethyl Laboratories), FLAG (2368, Cell Signaling), GST (27–4577–01, GE Healthcare), His (AM1010a, Abgent), hnRNP L (4D11, Abcam), p54^nrb/NONO (MA3–2024, Affinity Bioreagents), MATR3 (NB100–1761, Novus Biologicals), PSPC1 (a gift from Dr. Archa Fox).

Yarosh C.A., Tapescu I., Thompson M.G., Qiu J., Mallory M.J., Fu X.D, & Lynch K.W. (2015). TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells. Nucleic Acids Research, 43(18), 9006-9016.

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GABA Receptor Subunit Quantification

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Protein samples were lysed with SDS (2%), and their concentration was determined using BCA assays. Samples were separated using SDS-PAGE and transferred onto a nitrocellulose membrane, which was then incubated with His₆ tag antibody (1:1000; catalog no. AM1010a, Abgent) or the subunit-specific primary antibodies as follows: rabbit anti-GABA_AR γ2 subunit (1:1000; catalog no. 224003, Synaptic Systems); anti-GABA_AR β2/β3 subunit (1:50; UCL 112, raised against the N-terminal peptide of the β2 subunit in Dr. J. Jovanovic's laboratory); anti-GABA_AR β3 subunit (1:500, UCL 74 (47 (link))); anti-GABA_AR α1 subunit (1:500 (46 (link))); anti-GABA_AR α2 subunit (1:500, catalog no. 224102, Synaptic Systems), or anti-GABA_AR α2 subunit (2 μg/μl, raised against the intracellular loop (416–424) of the α2 subunit (48 (link))). Membranes were washed and incubated with the alkaline phosphatase-conjugated anti-rabbit secondary antibody (1:2500; catalog no. 31340, Invitrogen) or the HRP-conjugated anti-rabbit antibody (1:1000; catalog no. 711-035-152-Jackson ImmunoResearch). Immunoreactivity was detected using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium reagents or the SuperSignal West Femto Chemiluminescent substrate (catalog no. 34095, Fisher Scientific; Loughborough, UK), respectively.

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Exo70p-Bem1p Interaction Kinetics

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The K_d for the Exo70p–Bem1p interaction was determined as described previously (Pollard, 2010 (link)). In brief, 0–4.59 µM of GST-Exo70p, pre-immobilized on glutathione beads, was mixed with 25 nM Bem1p-His₆ in 200 µl of binding buffer (1× PBS, pH 7.4, 1 mM DTT, and 0.1% Triton X-100) and incubated at room temperature for 1 h. The binding mixture was centrifuged at 5,000 rpm for 1 min. The amount of Bem1p-His₆ remaining in the supernatant was determined by Western blotting using anti-His antibody (AM1010a; Abgent) and quantified with an Odyssey imaging system. The K_d was calculated with Origin software.

Liu D, & Novick P. (2014). Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p. The Journal of Cell Biology, 207(1), 59-72.

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Acylated Nucleosome Binding to BRD4

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Following sucrose gradient purification, chick erythrocyte nucleosomes were chemically acylated as previously described^{32 (link)} with 1 mM acetic, propionic, butyric or glutaric anhydride in the presence of 100 mM NaCl for 1 h at room temperature. Acylation reactions were quenched with 100 mM Tris (pH 8.0). Nucleosomes were precipitated by adding 1 reaction volume of 10% w/v trichloroacetic acid, pelleted by centrifugation at 15,000 rpm for 10 min, and resuspended in 25 mM Tris (pH 8.0) with 100 mM NaCl. 200 μL of mononucleosomes (500 nM) combined with 2.5 μM His₆-tagged BRD4-BD1 were applied to a 4 mL sucrose gradient (5–20% w/v sucrose with 1 mM EDTA, pH 8.0) and centrifuged at 55,000 × g for 3.5 h. The sucrose gradient was then collected in fractions. Nucleosome-containing fractions were identified by agarose gel electrophoresis combined with ethidium bromide staining and BRD4-BD1 containing fractions were identified by anti-His₆ tag immunoblotting. Membranes were blocked with PBST with 3% w/v BSA and western blots were performed using an anti-His₆ tag primary antibody (Abgent, AM1010A) at a dilution of 1:1000 followed by anti-mouse IgG secondary antibody HRP (GeneTex, GTX213111-01) at 1:10,000 and detection by chemiluminescence.

Olp M.D., Zhu N, & Smith B.C. (2017). Metabolically-derived lysine acylations and neighboring modifications tune BET bromodomain binding to histone H4. Biochemistry, 56(41), 5485-5495.

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Bem1-Exo70p Interaction Characterization

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GST-Exo70p WT, fragments, or mutants, pre-immobilized on glutathione beads, were incubated with purified C-terminal His₆-tagged WT Bem1, its fragments, or mutants, in binding buffer (1× PBS, pH 7.4, 1 mM DTT, 0.1% Triton X-100, and 1 mg/ml BSA). The total volume for each binding reaction was 200 µl. The final concentrations for GST-Exo70p and Bem1p-His₆ were 0.15 µM and 0.1 µM, respectively. The binding mixture was incubated at room temperature for 1 h followed by five washes with binding buffer without BSA. Bound proteins were resolved with Laemmli sample buffer and subjected to CBB staining to detect GST fusion proteins and Western blotting with mouse monoclonal anti-His antibody (AM1010a, 1:1,000; Abgent) to detect associated His₆-tagged proteins.

Liu D, & Novick P. (2014). Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p. The Journal of Cell Biology, 207(1), 59-72.

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