Ab9485

Manufactured by Merck Group

Sourced in United States, United Kingdom

Ab9485 is a laboratory instrument designed for the analysis and detection of biological samples. It is capable of performing various analytical techniques, such as spectroscopy and immunoassays. The core function of the Ab9485 is to provide researchers and scientists with a reliable and accurate tool for their scientific investigations.

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Lab products found in correlation

Ab2302, by Merck Group (2 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) Ripa protein extraction reagent, by Beyotime (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions) Puromycin, by Merck Group (1 mentions) Ab71978, by Abcam (1 mentions) A5441, by Merck Group (1 mentions) Ab151769, by Abcam (1 mentions) U6grna cas9 2a rfp, by Merck Group (1 mentions) Imagelab software version 4, by Bio-Rad (1 mentions) Ripa buffer, by Merck Group (1 mentions) Ab205718, by Merck Group (1 mentions) Ab38898, by Merck Group (1 mentions)

3 protocols using ab9485

Protein Extraction and Western Blot Analysis

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Using of RIPA protein extraction reagent (Beyotime, Shanghai, China) with PMSF (Roche, Basel, Switzerland) to lyse cells. Approximately 25 μg of protein extracts were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes (Sigma, St. Louis, MO, USA), and probed with primary antibodies (anti-ORC1, ab85830, 1:5000; anti-actived-caspase3, ab2302, 1 µg/mL; anti-Bcl-2, ab692, 1:500; GAPDH, ab9485, 1:2500). Subsequently, secondary antibodies were provided to culture the cells (anti-Rabbit IgG H&L, ab6721, 1:10,000; anti-Mouse IgG H&L, ab205719, 1:10,000; Abcam). Membranes were in incubation at 4 °C for 24 h, then with secondary antibodies for another 2 h. The bands were detected by enhanced chemiluminescent (ECL) method. All antibodies were brought from Abcam (Cambridge, MA, USA).

Chen X., Xiong D., Ye L., Wang K., Huang L., Mei S., Wu J., Chen S., Lai X., Zheng L, & Wang M. (2019). Up-regulated lncRNA XIST contributes to progression of cervical cancer via regulating miR-140-5p and ORC1. Cancer Cell International, 19, 45.

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CRISPR-Cas9 Knockout of CSE, SQR, and MST

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The following guide RNAs (gRNAs) were used in the study: For CSE: exon 5 (5’-GGAGACATTATTTTGGTCGTGG-3’) and exon 1 (5’-TTCCAACATTTGGCCACGCAGG-3’) (purchased from Sigma-Aldrich); for SQR: exon 8 (5’-GTCCTCAAGACCAGTCCTGTGG-3’) (Sigma-Aldrich) and exon 3 (5’-TGCCGTGGGACGACCAGATG-3’) (Genscript); for MST: exon 4 (5’-CGCGTTACCGTCTCGGGGCT-3’) (Genscript). The gRNAs were expressed using an U6gRNA-Cas9-2A-RFP (Sigma-Aldrich), or pSpCas9 BB-2A-Puro (PX459) v2.0 vector after transient transfection using Lipofectamine LTX following the manufacturer’s protocol. Depending on the vector, cells were selected either with 12.5 μg/ml puromycin (Sigma), or by FACS. Individual clones were grown after diluting cells to a concentration of 0.5 cells per 100 μl in 96 well plates. Knockout clones were identified by western blotting. Antibodies used were rabbit anti-CSE (abcam, ab151769) at a dilution of 1:500, mouse anti-SQR (abcam, ab71978) at a dilution of 1:500 and mouse anti-MST (Santa Cruz, sc-374326) at a dilution of 1:100. Rabbit anti-GAPDH (abcam, ab 9485) (1:10000) and mouse anti-β-actin (sigma # A5441) (1:2500) were used as loading controls.

Ezeriņa D., Takano Y., Hanaoka K., Urano Y, & Dick T.P. (2018). N-acetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur production. Cell chemical biology, 25(4), 447-459.e4.

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Protein Extraction and Western Blot Protocol

Cited 1 time

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Fresh protein lysis solution was produced by mixing RIPA Buffer (Sigma-Aldrich), protease inhibitor PMSF (Sangon), and phosphatase inhibitor Sodium orthovanadate (V) dodecahydrate (Sangon) with the ratio of 98:1:1 (v/v/v). Total proteins were extracted by adding the protein lysis solution for incubation on ice for 30 min and collecting the supernatant through centrifugation at 7000 rpm/min for 15 min. The operating steps for Western blot followed the detailed descriptions published previously.21 (link) The antibodies were all bought from Abcam (Cambridge, UK): anti-proliferating cell nuclear antigen (anti-PCNA; ab18197, 1:1000), anti-Cyclin D1 (ab226977, 1:1000), anti-total-caspse3 (anti-t-caspase 3; ab4051, 1:500), anti-cleaved caspase 3 (anti-C-caspase 3; ab2302, 1:1000), anti-matrix metalloproteinase 9 (anti-MMP9, ab38898, 1:1000), anti-NOVA2 (Sigma-Aldrich, AV40399, 1:1000), anti-GAPDH (ab9485, 1:1000) and Goat Anti-Rabbit IgG H&L (HRP) second antibody (ab205718, 1:3000). GAPDH acted as the internal control in this assay. ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA) was exploited to perform the grey level analysis.

Li G., Zhao C., Zhang H., Yu J., Sun Y, & Zhang Y. (2020). Hsa_circ_0046263 Drives the Carcinogenesis and Metastasis of Non-Small Cell Lung Cancer Through the Promotion of NOVA2 by Absorbing Mir-940 as a Molecular Sponge. Cancer Management and Research, 12, 12779-12790.

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