Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study

Abstract

Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin b-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin b-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.

Deletion of Small Ubiquitin-like Modifier-1 Attenuates Behavioral and Anatomical Deficits by Enhancing Functional Autophagic Activities in Huntington Disease

“…Western blotting analysis of striatal brain lysates prepared in non-denaturing Tris buffer (which enriches cytoplasmic proteins [121,122]) showed a significant enhancement of EM48+ aggregates in the Q175DN-SUMO1KO striatum, compared to the Q175DN striatum (Fig. 4K, L), supporting confocal findings that SUMO1 deletion increased the cytoplasmic EM48+ mHTT levels.…”

Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells

“…Proteins were extracted from cultured control and Dsg2 mut/mut cardiac- and BM-MSCs, using the lysis buffer, described in Muinao et al [ 20 ]. WB was then performed as described in Zaglia et al and Muinao et al [ 19 , 20 ]. Primary antibodies used in this study are listed in Supplementary Table S3 .…”

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study

“…To obtain protein extracts, trophozoites washed three times with Page's Saline Solution (1 mmol L −1 Na 2 HPO 4 , 1 mmol L −1 KH 2 PO 4 , 0.016 mmol L −1 MgSO 4 , 0.03 mmol L −1 CaCl 2 and 2 mmol L −1 NaCl) (400 × g , 5 min) were incubated for 1 h at 4 °C in lysis buffer (10 mmol L −1 Tris-HCl, pH 7.6, 50 mmol L −1 NaCl, 50 mmol L −1 NaF, 1% Triton X-100 and Halt ™ Protease Inhibitor Cocktail, Thermo Scientific) (Muinao et al ., 2018). The suspension was centrifuged at 3000 × g for 20 min and the protein concentration of the soluble fraction was determined by Bradford assay, taking into account proper dilution of the sample to avoid interference from any of the lysis buffer components.…”