Search
Overview of protein extraction from cells and tissues
The study of proteins in living organisms is an integral part of life science research. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function.
The first step in protein analysis is cellular extraction. Because proteins are so heterogeneous, there is no one method or reagent that is optimal for general protein isolation. In addition, protein extraction techniques vary depending on the source of the starting material, the location within the cell of the protein of interest and the downstream application.
Many techniques have been developed to obtain the best protein yield and purity for different types of cells and tissues, taking into account where appropriate, the subcellular location of the protein and the compatibility of the protein extract with the next step in the experiment.
Key considerations for protein extraction
- Sample type
- Protein location
- Protein Stabilisation During Extraction
In life science research, proteins are typically extracted from cultured mammalian cells, mammalian tissues or primary cells.
When extracting proteins from mammalian tissues, mechanical disruption is required to isolate the cells from their tissue matrix. For cultured mammalian and primary cells, which have only a plasma membrane separating the cell contents from the environment, reagents containing detergents and other components can easily disrupt the protein-lipid membrane bilayer, making total protein extraction relatively straightforward.
Other organisms that are also commonly used in protein research, including bacteria (as a tool for protein expression), yeast (as a model for cell biology) and plants (for agricultural biotechnology) contain cell walls, which have traditionally required mechanical lysis. However, detergent-based solutions have been developed to efficiently lyse these cells without using physical disruption. Table 1 describes the most common methods for protein extraction, as well as the advantages and disadvantages of each method.
Methods used for cell or tissue disruption
| Lysis Method | Apparatus |
Method |
Sample Type | Fractions Isolated | Advantages | Disadvantages |
Reagent based |
None, except for tissues | Disruption of lipid membrane and/or cell wall | Mammalian, bacterial, yeast, insect, and plant cells and /or tissues |
Total protein or subcellular fractions or organelles | Rapid, gentle, efficient, reproducible, high protein yield | Some components may need to be removed for downstream analysis |
|
Waring blender |
Shearing caused by rotating blades |
Mammalian tissues or cells |
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur, noisy |
|
Polytron mixer |
Shearing occurs within long shaft containing rotating blades |
Mammalian tissues or cells
|
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur |
|
Bead beater |
Disruption caused by collision with agitated beads in liquid suspension |
Mammalian tissues or cells, yeast or bacterial cells, plant tissue |
Total protein | Low shearing, works with wide range of cell and tissue types, good for hard, difficult tissues |
Denaturing may occur due to heat generated, noisy |
Mechanical |
Dounce homogeniser |
Shearing caused by a round glass pestle that is manually driven into a glass tube |
Mammalian tissues or cells |
Total protein, mitochondria |
Inexpensive equipment, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
Potter-Elvehjem |
Shearing is caused by a PTFE- coated pestle that is manually or mechanically driven into a conical vessel |
Mammalian tissues or cells |
Total protein | Hand-held device, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
|
| French press | Shearing is caused by high pressure when sample is forced through a small hole |
Bacterial cells | Total protein | Rapid, efficient | Minimum 40mL samples, very expensive, noisy, lengthy set-up and clean-up |
|
| Sonication | Sonicator | Shearing is caused by high frequency sound waves |
Mammalian tissues or cells, yeast or bacterial cells |
Total protein | Directly compatible with downstream applications |
Reproducibility may vary, denaturing may occur, noisy |
Freeze/ Thaw |
Freezer or dry ice/ ethanol in container |
Disruption is caused by the formation of ice crystalsin membranes |
Bacterial and mammalian cells |
Total protein | Rapid, gentle, efficient, reproducible, high protein yield |
Some components may need to be removed for downstream analysis |
| ManualGrinding | Mortar and pestle |
Disruption is caused by the formation of ice crystals in membranes |
Plant tissue | Total protein | Inexpensive | Reproducibility may vary, laborious |
Because certain proteins are localized in specific organelles, protein yield and enrichment are greatly improved if the protein is extracted directly from its subcellular compartment or organelle. Some types of mechanical lysis alone can disrupt all cellular compartments, making it difficult to achieve subcellular fractionation.
However, by the careful optimisation of physical disruption and detergent-buffer formulations, procedures have been developed that enable the separation of subcellular structures. For example, with the appropriate detergents, hydrophobic membrane proteins can be solubilised and separated from hydrophilic proteins. A combination of tools and steps enables the isolation of intact nuclei, mitochondria and other organelles for study or protein solubilisation.
Methods used for cell or tissue disruption
| Lysis Method | Apparatus |
Method |
Sample Type | Fractions Isolated | Advantages | Disadvantages |
Reagent based |
None, except for tissues | Disruption of lipid membrane and/or cell wall | Mammalian, bacterial, yeast, insect, and plant cells and /or tissues |
Total protein or subcellular fractions or organelles | Rapid, gentle, efficient, reproducible, high protein yield | Some components may need to be removed for downstream analysis |
|
Waring blender |
Shearing caused by rotating blades |
Mammalian tissues or cells |
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur, noisy |
|
Polytron mixer |
Shearing occurs within long shaft containing rotating blades |
Mammalian tissues or cells
|
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur |
|
Bead beater |
Disruption caused by collision with agitated beads in liquid suspension |
Mammalian tissues or cells, yeast or bacterial cells, plant tissue |
Total protein | Low shearing, works with wide range of cell and tissue types, good for hard, difficult tissues |
Denaturing may occur due to heat generated, noisy |
Mechanical |
Dounce homogenizer |
Shearing caused by a round glass pestle that is manually driven into a glass tube |
Mammalian tissues or cells |
Total protein, mitochondria |
Inexpensive equipment, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
Potter-Elvehjem |
Shearing is caused by a PTFE- coated pestle that is manually or mechanically driven into a conical vessel |
Mammalian tissues or cells |
Total protein | Hand-held device, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
|
| French press | Shearing is caused by high pressure when sample is forced through a small hole |
Bacterial cells | Total protein | Rapid, efficient | Minimum 40mL samples, very expensive, noisy, lengthy set-up and clean-up |
|
| Sonication | Sonicator | Shearing is caused by high frequency sound waves |
Mammalian tissues or cells, yeast or bacterial cells |
Total protein | Directly compatible with downstream applications |
Reproducibility may vary, denaturing may occur, noisy |
Freeze/ Thaw |
Freezer or dry ice/ ethanol in container |
Disruption is caused by the formation of ice crystalsin membranes |
Bacterial and mammalian cells |
Total protein | Rapid, gentle, efficient, reproducible, high protein yield |
Some components may need to be removed for downstream analysis |
| ManualGrinding | Mortar and pestle |
Disruption is caused by the formation of ice crystals in membranes |
Plant tissue | Total protein | Inexpensive | Reproducibility may vary, labori o us |
Cell lysis disrupts cell membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent degradation of extracted proteins and obtain the best possible protein yield and activity following cell lysis, protease and phosphatase inhibitors can be added to the lysis reagents.
Numerous compounds have been identified and used to inactivate or block the activities of proteases and phosphatases by reversibly or irreversibly binding to them. Because some detergents used in protein extraction formulations may inactivate the function of enzymes of interest or affect their long-term stability, it may be important to remove the detergents following cell lysis.
In addition, high concentrations of detergents or salts can interfere with common research methods such as protein assays, protein purification, immunoprecipitation, gel electrophoresis and mass spectrometry (MS). In some cases, interfering substances can be mitigated simply by dilution or dialysis.
Methods used for cell or tissue disruption
| Lysis Method | Apparatus |
Method |
Sample Type | Fractions Isolated | Advantages | Disadvantages |
Reagent based |
None, except for tissues | Disruption of lipid membrane and/or cell wall | Mammalian, bacterial, yeast, insect, and plant cells and /or tissues |
Total protein or subcellular fractions or organelles | Rapid, gentle, efficient, reproducible, high protein yield | Some components may need to be removed for downstream analysis |
|
Waring blender |
Shearing caused by rotating blades |
Mammalian tissues or cells |
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur, noisy |
|
Polytron mixer |
Shearing occurs within long shaft containing rotating blades |
Mammalian tissues or cells
|
Total protein | Inexpensive equipment |
Reproducibility may vary, denaturing may occur |
|
Bead beater |
Disruption caused by collision with agitated beads in liquid suspension |
Mammalian tissues or cells, yeast or bacterial cells, plant tissue |
Total protein | Low shearing, works with wide range of cell and tissue types, good for hard, difficult tissues |
Denaturing may occur due to heat generated, noisy |
Mechanical |
Dounce homogenizer |
Shearing caused by a round glass pestle that is manually driven into a glass tube |
Mammalian tissues or cells |
Total protein, mitochondria |
Inexpensive equipment, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
Potter-Elvehjem |
Shearing is caused by a PTFE- coated pestle that is manually or mechanically driven into a conical vessel |
Mammalian tissues or cells |
Total protein | Hand-held device, ideal for small volumes |
Reproducibility may vary, denaturing may occur |
|
| French press | Shearing is caused by high pressure when sample is forced through a small hole |
Bacterial cells | Total protein | Rapid, efficient | Minimum 40mL samples, very expensive, noisy, lengthy set-up and clean-up |
|
| Sonication | Sonicator | Shearing is caused by high frequency sound waves |
Mammalian tissues or cells, yeast or bacterial cells |
Total protein | Directly compatible with downstream applications |
Reproducibility may vary, denaturing may occur, noisy |
Freeze/ Thaw |
Freezer or dry ice/ ethanol in container |
Disruption is caused by the formation of ice crystalsin membranes |
Bacterial and mammalian cells |
Total protein | Rapid, gentle, efficient, reproducible, high protein yield |
Some components may need to be removed for downstream analysis |
| ManualGrinding | Mortar and pestle |
Disruption is caused by the formation of ice crystals in membranes |
Plant tissue | Total protein | Inexpensive | Reproducibility may vary, labori o us |
Cell Lysis
- Recovery of large molecular weight proteins (high MW proteins)
- Improved cell lysis and DNA digestion, thereby releasing soluble proteins and reducing viscosity to increase yields
- Detergent in Tris buffer with Lysozyme and Universal Nuclease
- Cost-effective equivalent to Merck Millipore Bugbuster™ Master Mix
Ordering Information
- Gentle
Mild detergent lysis, yielding extracts that are immediately compatible with coomassie (Bradford) and BCA protein assays or SDS-PAGE - Compatible
Extracts soluble proteins in non-denatured state, enabling direct use in immunoprecipitation and other affinity purification procedures - Amine-free and dialysable
Formulation ensures compatibility with subsequent assay systems - Convenient
Lyse adherent cells directly in plate or after scraping and washing in suspension - Non-denaturing
Maintain activity of luciferase, beta-galactosidase, CAT and other reporter genes as well
Ordering Information
- Fast
Obtain nuclear and cytoplasmic fractions in less than two hours - Proven
The NE-PER Reagent Kit is referenced in more than 950 distinct publications - Scalable
Kit sizes for producing extracts from cells and tissues - Convenient
Simple instructions do not require ultracentrifugation over gradients - Compatible
Use for downstream assays, including western blotting, gel-shift assays, protein assays, reporter gene assays and enzyme activity assays
Ordering Information
- Concise
Extract functional cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeletal protein fractions in less than 3 hours - Convenient
Perform a simple procedure without using ultracentrifugation over gradients - Complete
Obtain cytoplasmic, membrane, soluble nuclear, chromatin-bound and cytoskeletal protein fractions from a single kit - Compatible
Use extracts for downstream applications such as protein assays, western blotting, gel-shift assays and enzyme activity assays - Versatile
Pick the kit to extract from cultured cells or tissues
Ordering Information
- Ease-of-use
All-in-one formulation to extract and stabilise GPCRs in one hour - GPCR preservation
Stabilises the receptor in a detergent micelle to ensure receptor function - Optimised
Formulation and procedure provide gentle but efficient extraction of GPCRs from the plasma membrane - Compatible
Simple instructions do not require ultracentrifugation over gradients - Compatible
Lysate is directly compatible with downstream applications including western blotting, immunoprecipitation, and receptor ligand binding assays
Ordering Information
Inhibitors
The inhibitor cocktails are available as both 100X cocktail solutions (i.e., Thermo Scientific Halt Inhibitor Cocktails) and quick dissolving tablets (Thermo Scientific™ Inhibitor Tablets) to accommodate general and specific needs in cell lysis and protein extraction methods. These inhibitors are ideal for the protection of proteins during extraction or lysate preparation from cultured cells, animal tissues, plant tissues, yeast or bacteria. All Halt Inhibitor Cocktails and Inhibitor Tablets are compatible with Thermo Scientific Protein Extraction Reagents and most homemade and commercial cell lysis solutions.
- Multiple package sizes
Liquid cocktails are available in 100 μL single use format or 1, 5 and 10 mL pack sizes; tablets come in two sizes—for 10 or 50 mL volumes to accommodate different volume/pricing needs. - Convenient
The refrigerator-stable, 100X liquid or tablet format is more effective and easier to use than individual inhibitors; just pipette the amount you need, or add a tablet to a 10 or 50 mL solution - No proprietary ingredients
Fully disclosed formulation - Two popular formulations
Available with or without EDTA; EDTA-free formulation ensures compatibility with isoelectric focusing or His-tag purification - Complete protection
All-in-one formulations contain both protease and phosphatase inhibitors (combined cocktail only) - Compatible
Use with Thermo Scientific™ Cell Lysis Buffers or nearly any other commercial or homemade detergent-based lysis reagent; also works with standard protein assays, including BCA and coomassie (Bradford)
Components present in the Thermo Scientific Halt Inhibitor Cocktails and Thermo Scientific Protease and Phosphatase Inhibitor Tablets
Inhibitor component |
Target (mechanism) | Protease liquid cocktails and tablets |
Phosphatase liquid cocktails and tablets |
Combined protease and phosphatase liquid cocktails and tablets |
|
|---|---|---|---|---|---|
| AEBSF•HC | Serine Proteases (irreversible) | X | |||
| Aprotinin Serine | Protease (reversible) | X | X | ||
| Bestatin | Aminopeptidase (reversible) | X | X | ||
| E-64 | Cysteine (irreversible) | X | X | ||
| E-64 | Serine and Cysteine Protease (reversible) |
X | X | X | |
| Pepstatin | Aspartic acid proteases (reversible) |
X | |||
| EDTA† | Metalloproteases (reversible) | X | X | ||
| Sodium Fluoride | Serine-Threonine and Acidic Phosphatases |
X | X | ||
| Sodium Orthovanadate | Tyrosine and Alkaline Phosphatases |
X | X | ||
| β-glycero-phosphate | Serine-Threonine Phosphatase | X | X | ||
| Sodium Pyrophosphate | Serine-Threonine Phosphatase | X | X |
† EDTA not in EDTA-free formulations.
Liquids
Protease Inhibitor Cocktails
Phosphatase Inhibitor Cocktails
Combined Protease and Phosphatase Inhibitors Cocktails
Tablets
Protease Inhibitor Tablets
Phosphatase Inhibitor Tablets
Combines Protease and Phosphatase Inhibitor Tablets
Dialysis and Desalting
- Convenient
Pipette-accessible for easy sample loading and retrieval - Easy to use
Self-floating chambers for buoyancy and vertical orientation during dialysis - Sturdy construction
Ensures the highest possible sample integrity and protection - Fast and consistent
Maximum sample recovery - Versatile
Ideal for removing low molecular weight contaminants, performing buffer exchange and desalting - Gamma-irradiated option
Gamma-irradiated cassettes are available for applications requiring conditions that minimize the risk of sample contamination - Green
Up to 68% less plastic use vs. previous generation
Ordering Information
2K MWCO
3.5K MWCO
10K MWCO
10K MWCO—Gamma Irradiated
20K MWCO
- High performance
Exceptional protein recovery and minimal sample dilution - Fast
No screening fractions for protein or waiting for protein to emerge by gravity-flow - Flexible
A product offering to accommodate samples from 2 μL to 4 mL - Easy to use
Avoid use of cumbersome column preparation or equilibration
| Column sizes and recommended sample volumes | ||
|---|---|---|
Column size |
Resin bed | Sample size |
| Micro | 75 μL | 2–12 μL |
| 0.5 mL | 0.5 mL | 30–130 μL |
| 2 mL | 2 mL | 200–700 μL |
| 5 mL | 5 mL | 500–2,000 μL |
| 10 mL | 10 mL |
1,500–4,000 μL |
For Research Use Only. Not for use in diagnostic procedures.