Mammalian tissues are highly complex and contain a diverse range of proteins with varying properties, making the preparation of high-quality protein extracts a critical step for successful two-dimensional electrophoresis (2-DE) analysis.
Tissue homogenization is the first and most important step in the preparation of mammalian tissue samples for 2-DE. The choice of homogenization method depends on the type of tissue and the properties of the proteins to be extracted. Mechanical disruption using a homogenizer or blender is often used for solid tissues, whereas enzymatic digestion using proteases is preferred for soft tissues.
Once the tissue is homogenized, protein extraction is typically performed using a buffer that contains detergents and chaotropes. Detergents help to solubilize membrane-bound proteins, while chaotropes disrupt protein-protein interactions and aid in protein denaturation. The choice of extraction buffer depends on the specific proteins of interest and their physicochemical properties.
Protein extracts may contain impurities that can interfere with downstream analysis, such as salts, lipids, and nucleic acids. Therefore, sample clean-up is often necessary to remove these contaminants and improve protein purity. Common clean-up methods include acetone precipitation, TCA/acetone precipitation, and cleanup with commercial kits.
After the protein extract is purified and solubilized, it can be subjected to 2-DE analysis. During 2-DE, proteins are first separated by isoelectric point using isoelectric focusing (IEF), followed by separation by molecular weight using SDS-PAGE. The separated proteins can then be visualized by staining with a protein dye, such as Coomassie Blue or silver stain.
To identify the proteins on the gel, mass spectrometry (MS) analysis is commonly used. Tandem MS (MS/MS) is often performed to obtain peptide sequences that can be used to search protein databases for protein identification.
1 Fresh Tissue
a) Fresh tissue should be stripped of connective tissue and fat. Preferably, 250 mg of tissue should be perfused with ice-cold saline prior to excision or at least briefly rinsed after excision and placed in a 50 ml beaker at room temperature (RT). Quickly add 8 volumes of lysis buffer (RT) to the tissue and mince thoroughly with surgical scissors. Cooling the urea-based lysis buffer will result in urea crystallization and is not recommended.
b) The minced tissue-sample slurry is then placed in a 3- or 5-mL DUALL ground-glass tissue grinder and manually homogenized. The duration and extent of manual homogenization depends on the connective-tissue content of the sample and should be continued until a uniform homogenate is formed and no tissue pieces are visible. Motor-driven homogenization should be avoided, as rapid pestle-rotation in ground glass will result in significant sample heating and protein carbamylation.
c) The resulting 12.5% homogenate is then allowed to remain at RT for 120 min, after which the samples are placed in Beckman polyallomer centrifuge tubes (1/2 × 2 in) and centrifuged at 100,000g for 30 min at 22°C using a Beckman TL-100 ultracentrifuge to remove nucleic acid and insoluble materials.
d) The clarified supernatant should be dispensed into small volumes and can be stored at -45°C or -80°C. Although tissue protein content (mg/g wet weight) may vary considerably among mammalian tissue types, the resulting protein concentration should be approximately 15-20 mg/mL. Tissue fluid abnormalities due to experimental conditions (e.g., edema, dehydration, etc.) can alter this estimate, so it is desirable to measure the protein concentration in dissolved samples. This can be done with any of a number of commercially available urea/detergent compatible protein assays such as the Bradford method (Pierce), RC DC Protein Assay (BioRad), and Noninterfering Protein Assay™ (Genotech). Keep in mind that their linear detection ranges are quite narrow.
2 Cultured Cells
a) The culture medium is first removed from the multi-well plate by aspiration. 400 μL of lysis buffer A or B (see Notes 2 and 3) is added directly to each well.
b) The culture plates are then placed in a 37°C incubator for 1 h with intermittent (every 15 min) manual agitation.
c) Following the 1-h solubilization, the entire volume is removed from each well and placed in 2-mL Eppendorf tubes.
d) Each sample is then sonicated with a Fisher Sonic Dismembranator using 3 × 2-s bursts at instrument setting #3. Sonication is conducted every 15 min for 1 h. Avoid contacting the sides of the tube with the sonicator probe as this generates bubbles/suds and potential sample loss.
e) At this point, samples may be ultracentrifuged (as above), after which the solubilized samples are transferred to microcentrifuge tubes for storage at –45°C or –80°C until thawed for analysis.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.