What is the source of the tissues in your products?

We obtain patient specimens from several established hospital-based collection sites around the world. Patients are informed and have consented to let their tissues be used for scientific study. They are neither compensated nor are their identities disclosed to us. We do however have clinical diagnosis and a detailed histopathology description of the tissues from board-certified pathologists which we provide to our clients.

What measures do you employ to ensure that the tissues are intact and that you minimize autolysis and protein degradation?

Every reasonable attempt is made to harvest and preserve tissue specimens quickly. The majority of our tissues are flash frozen within 5-10' of removal and then stored in liquid nitrogen until processing into lysates. Freezing tissues quickly after removal is significant because it has been shown that tissue ischemia causes the differential expression of 10-15% of all genes within 15' of tissue resection and that 30% of all proteins undergo significant changes in abundance within 30'. (Spruessel, A. et al., 2004. Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision. BioTechniques 36(6): 1030-1037.)

How are protein extracts prepared?

Frozen tissue is minced and homogenized in cold modified RIPA buffer [PBS (pH 7.4), 0.25% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 �g/ml Aprotinin, 1 �g/ml Leupeptin, 1 �g/ml Pepstatin A). Based on numerous mammalian tissue isolation protocols reported in the scientific literature and protocol manuals, we decided to use the average, recommended 1:9 (w/v) ratio of tissue to protein extraction buffer for maximize overall protein recovery. The first step involves mechanical shearing with a Polytron-type homogenizer at 35,000 rpm until the tissue is dispersed. The tissue solution is further disrupted by liquid shearing in a Dounce or Potter-Elvehjem homogenizer. This solution is centrifuged (20,000 x g; 20'; 4 degrees C) which is adequate to pellet most insoluble proteins, extra-cellular matrix, any intact nuclei, lysosomes and mitochondria. The supernatatant, which contains soluble proteins (cytosolic, nuclear and membrane) proteins, ER membranes and ribosomes, is saved. This fraction is formulated into 'soluble fraction' lysate and reverse protein microarray products. The resulting pellet is subjected to dissolution in Urea/Thiourea buffer using mechanical and liquid shearing. This protein solution is also centrifuged as above but at 15 degrees C to keep the salts from crystallizing. The supernatant from this step is formulated into 'insoluble fraction' lysate and reverse protein microarray products.

How are protein extracts quality controlled?

Protein solutions are quantitated using the Bio-Rad Protein Assay and plotted against standardized BSA and IgG protein solutions. Lysates are also routinely run on SDS-PAGE to assess protein quantity and quality.

Are there significant differences in proteins from normal vs. diseased tissues and/or between patients?

There exists a large potential bias in specific protein expression profiles due to tissue heterogeneity in and between normal and diseased tissue from different human patients. For example, tumor activity and structure can vary significantly between patients even though the cancers are classified and may be graded identically. Additionally, extraction conditions are not necessarily optimized for enrichment of proteins from specific families or classes. Instead, a widely accepted set of buffers is employed to fractionate soluble (modified RIPA buffer) and insoluble (Urea-based buffer) proteins. Expect some variation from materials prepared from different patients.