Cellular and subcellular extracts are used in a wide range of biochemical and proteomics studies to investigate the properties and functions of proteins within cells. These extracts can be obtained by various methods, depending on the type of cell or tissue and the protein of interest.
The first step in preparing cellular and subcellular extracts is cell lysis. This can be achieved by mechanical disruption, such as sonication or grinding, or by chemical lysis using detergents or chaotropic agents. The choice of lysis method depends on the type of cell or tissue and the properties of the proteins to be extracted.
Once the cells are lysed, the protein extract can be fractionated into different subcellular compartments, such as the cytoplasm, nucleus, mitochondria, and endoplasmic reticulum, by centrifugation or other separation methods. The choice of fractionation method depends on the properties of the proteins of interest and the downstream applications.
To further purify the protein extract, different chromatography techniques can be used, such as ion-exchange chromatography, affinity chromatography, and size-exclusion chromatography. These methods can separate proteins based on their charge, size, binding properties, or other characteristics.
After the protein extract is purified, it can be analyzed by various techniques, such as gel electrophoresis, western blotting, mass spectrometry, or enzymatic assays. These techniques can provide information on the identity, quantity, and function of the proteins in the extract.
1. Preparation of Protein Extracts from Mammalian Tissues and Cells
The preparation of protein extracts from many animal tissues is relatively simple because the cell membranes are weak and easily disrupted by osmotic and mechanical forces.
The usual first step in extraction is washing followed by homogenization in an appropriate buffer with a homogenizer and clarification by centrifugation. Centrifugation separates soluble proteins from membrane fractions and insoluble cell debris. Various cell suspensions can be prepared from tissues or organs either by mechanical methods or by enzymatic digestion. In general, enzymatic digestion is preferable because it is less damaging to cellular integrity. Alternatively, eth-ylene diamine tetracetic acid (EDTA) can usually be added to chelate Ca2+ ions, and calcium ions are often involved in cell-cell adhesion.
2. Protein Extraction from Cultured Cells
Extraction of proteins from cell lysis products is a prerequisite for proteomic analysis. Protein extraction can be performed by one-step extraction or by distribution extraction. One-step extraction method requires complete solubilization of cellular proteins (especially hydrophobic proteins), which may affect the comprehensiveness and accuracy of proteomic analysis. The stepwise extraction method not only improves the extraction rate of hydrophobic proteins, but also increases the resolution when separated by two-dimensional gel electrophoresis.
2.1 Cell culture and collection
Cells were cultured in specific culture medium [e.g. COS7 cell line was cultured in Dulbecco's Modified Engly Media (DMEM) medium containing 10% serum]. After the cells are cultured to logarithmic growth phase, they are digested with 0.25% trypsin and then centrifuged. Resuspend in the culture medium. Count and divide into two, centrifuge, discard culture medium, and wash 3 times with low salt phosphate buffer.
2.2 Stepwise extraction
Stepwise extraction is a three-step extraction with three lysis solutions suitable for solubilizing high, medium and low hydrophilic proteins, respectively.
Step 1: Extraction by lysis in aqueous solution. About 5 ~15 ng (wet weight) of cells were added to 2 ml of lysis solution I [40 mmol/L tris-aminomethane (Tris - base) pH 8.3 ~ 8.4]. Freeze and thaw repeatedly for 3~4 cycles. Add 20ug/ml bovine pancreatic deoxyribonuclease (DNase I) and 5ug/ml ribonuclease A (RNase A) at 4°C for 15 min and then shake and mix for 5 min. Centrifuge at 10°C at 12000r/min for 10 min. The supernatant was collected and freeze-dried. The remaining precipitate was washed twice with 40 mmol/L tris(hydroxymethyl)aminomethane solution.
Step 2: Extraction of lysate containing various components such as urea. To the Eppendrof microcentrifuge tube containing the precipitate from the previous step, 500 ul of lysate II ( 8 mol/L urea, 4% 3- [ (3-cholamidopropyl)-diethylamino]propanesulfonic acid (CHAPS), 100 mmol/L dithiotheietol (DTT), 40 mmol/L Tris- base and 0.5% pharmaltes pH 3 ~ 10, 20ug/ml DNase I and 5ug/ml RNase A). Mix with strong shaking for 5 min and then centrifuge at 10°C for 60 min at 14000 r/min. The supernatant was collected. The remaining precipitate was washed twice with 40 mmol/L Tris - base solution.
Step 3: Extraction with lysate containing thiourea-octanoylthioglycine trimethylene salt-tributyl phosphorus. In an Eppendrof microcentrifuge tube containing the precipitate from the previous step, 200 ul of lysate III (5 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 2% caprylyl sulfobetain (SB3-10), 2 mmol/L tributyl phosphine (TBP), 40 mmol/L Tris-base and 0.5% ph phosphine (TBP), 40 mmol/L Tris-base and 0.5% pharmaltes, pH 3 ~ 10, 20ug/ml DNase I and 5ug/ml RNase A). After mixing with strong shaking for 5 min, centrifuge at 10°C for 10 min at 12000 r/ min. The supernatant was collected.
3. Preparation of subcellular extracts
There is no single good method for the graded isolation of all tissues. It cannot be envisaged that the graded separation used for one tissue can be applied to other tissues. There are two main steps in the process of subcellular graded separation: homogenization of tissues and cells and separation of organelles.
The basic aim of any homogenization is maximum cell disruption. Destructive forces are used to cause minimal damage to the organelles of interest (e.g. protein denaturation and enzymatic degradation) as well as to maintain the original structural and functional integrity of the organelles of interest.
The basic methods of cell disruption are: ultrasonic vibration, mechanical grinding or shearing and nitrogencavitation. Methods based on the physical properties of the subcellular fractions are then applied for separation.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.