To detect protein interactions in cells, a Duolink® PLA in-situ kit (92101; Sigma–Aldrich Corp.) was used according to the manufacturer’s instructions. The primary antibodies were as follows: a rabbit anti-UBA52 antibody (EPR4547; ab109230; Abcam) and a mouse anti-CDK6 antibody (ab54576; Abcam). The primary antibody rabbit anti-Immunoglobulin G was used as a control (ab125938; Abcam). Images were acquired with a confocal laser microscope using a ×60 oil immersion objective lens. Dots within cells were counted using Duolink ImageTool (OLINK Bioscience, Uppsala, Sweden). Cells were only counted when the whole cell was in the field of vision.
Ab54576
Manufactured by Abcam
Ab54576 is a lab equipment product offered by Abcam. It is a high-quality instrument designed for use in scientific research applications. The core function of this product is to perform a specific task required in the laboratory setting. Detailed information about its intended use or features is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab125938, by Abcam (1 mentions)
Duolink in situ pla kit, by Merck Group (1 mentions)
Duolink image tool, by Olink (1 mentions)
Ab199728, by Abcam (1 mentions)
Ab184796, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Pdvf membranes, by Roche (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab9485, by Abcam (1 mentions)
2 protocols using ab54576
1
Protein Interaction Analysis via Duolink PLA
2
Western Blot Analysis of Cell Cycle Regulators
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Cells were resuspended in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4). Cell lysates were incubated on ice for 20 mins and supernatants were collected after high-speed centrifuge at 4°C. Protein concentration was measured by Bradford assay. Samples were loaded evenly into SDS-PAGE and transferred onto PDVF membranes (Roche). Membranes were blocked in 5% skim milk at room temperature and then incubated with primary antibodies overnight at 4°C. After washing with PBS-T (PBS solution, 0.1% Tween-20) and incubation of secondary antibodies, membranes were incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific) and protein bands were developed in dark room. Antibodies used for Western blot assay were: CCND1 (abcam, ab16663), pRb (abcam, ab184796), CDK4 (abcam, ab199728), CDK6 (abcam, ab54576), GAPDH (abcam, ab9485).
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