Protein Extraction from Tissue Samples

The tissue and tumor samples were homogenized with a Bullet Blender (Next Advance, Averill Park, NY) according to the manufacturer’s instructions. Sequential extractions of frozen samples of tumors were performed using the CNMCS compartmental protein extraction kit (Millipore, Billerica, MA) as previously described [13 (link)]. In brief, frozen samples were homogenized and extracted sequentially to remove preferentially (1) cytosolic proteins, (2) nuclear proteins, (3) membrane proteins, (4) cytoskeletal proteins leaving a final insoluble fraction enriched for ECM proteins, although different tissues behave somewhat differently in the ease of extraction so that proteins sometimes appear in more than one fraction. The effectiveness of extraction of specific proteins was monitored by immunoblotting using the following antibodies: rabbit anti-collagen I, mouse anti-GAPDH and rabbit anti-histones (Millipore, Billerica, MA), the rabbit anti-actin antibody (serum 14–1) was generated in our laboratory.

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Naba A., Clauser K.R., Whittaker C.A., Carr S.A., Tanabe K.K, & Hynes R.O. (2014). Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver. BMC Cancer, 14, 518.

Publication 2014

Bullet Blender CNMCS compartmental protein extraction kit mouse anti-GAPDH rabbit anti-histones

Actin Anti antibody Antibodies Collagen i Cytoskeletal proteins Cytosolic Ecm proteins Frozen Gapdh Histones Membrane proteins Mouse Nuclear proteins Proteins Rabbit Serum Tissues Tumors

Corresponding Organization :

Other organizations : Massachusetts Institute of Technology, Broad Institute, Massachusetts General Hospital, Howard Hughes Medical Institute

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Variable analysis

independent variables

Tissue and tumor samples homogenization method (Bullet Blender)
Sequential protein extraction method (CNMCS compartmental protein extraction kit)

dependent variables

Extraction of cytosolic, nuclear, membrane, and cytoskeletal proteins
Enrichment of extracellular matrix (ECM) proteins in the final insoluble fraction

control variables

Frozen tissue and tumor samples
Manufacturer's instructions for Bullet Blender
Previously described protocol for CNMCS compartmental protein extraction kit [13]

controls

Positive controls: Immunoblotting with antibodies against collagen I, GAPDH, and histones
Negative control: Not explicitly mentioned

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