Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

Abstract

Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE). This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer

“…Protein lysates were separated by 10% sodium lauryl sulphate polyacrylamide (SDS‐PAGE) gel, and then transferred onto apolyvinylidene difluoride (PVDF) membrane (Sigma‐Aldrich). The membranes were blocked andincubated at 4°C overnight with either an anti‐EpcamAb (R&D systems, MAB960) or an anti‐tsg101 Ab (Abcam, ab1337586), as tsg101 is reported to be widely expressed in EVs and could be used for normalization [19,20]. After being incubated with a secondary antibody (anti‐mouse IgG (HAF007, R&D systems) for anti‐Epcam, anti‐rabbit IgG (ab205718, Abcam) for anti‐tsg101) for 1 h at room temperature, the signals were visualized using the LI‐COR Image Studio lite imaging system.…”

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice

“…Proteins were extracted with frozen tumor tissues with the use of protocols modified from Zakharchenko et al 42 Briefly, 10 to 20 mg of frozen tissue was homogenized in 250 mL of RIPA buffer (50 mmol/L Tris-HCl pH 7.5, 105 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and 2 mmol/L EDTA) with protease inhibitors (catalog number 78439; Thermo Fisher Scientific, Waltham, MA) and phosphatase inhibitors (catalog number 78445; Thermo Fisher Scientific) with the use of an electric drill with a plastic pellet pestle (catalog number 7495150000; Kimble-Chase) attached. Homogenized lysates were incubated on ice for 30 minutes, mixed with a vortex mixer, and briefly sonicated.…”

Humoral immunoprofiling identifies novel biomarkers and an immune suppressive autoantibody phenotype at the site of disease in pancreatic ductal adenocarcinoma

“…Tissue matched from a subset of PDAC patients in the serum cohort was collected for assessment of local autoantibody production. Here, paired tissue sections of ~3mm 2 for PDAC tumour (n=8), normal adjacent (n=8), and chronic pancreatitis (n=8) were lysed for antibody extraction as previously described ( 26 ) and antibody presence in tissue lysates was confirmed by an immunoglobulin affinity purification method using magnetic Protein A and Protein G microbeads (MagReSyn®), as per to the manufacturer’s protocol, and as previously described ( 27 , 28 ) Serum and tissue samples were stored at -80 until assays were performed.…”