The Protein Man's Blog | A Discussion of Protein Research

What should we consider when selecting a protein extraction buffer?

Protein extraction buffers are complex and may contain reducing agents, detergents, salts, imidazole, chelators, protease inhibitors, osmolytes, co-factors, and stabilizers. These chemicals maximize protein yield while maintaining integrity and functionality.

Protein extraction buffers are designed very carefully to achieve three critical objectives in protein experiments:

1. They ensure that the protein purification procedure yields high yields of the target protein and that the protein is not degraded during the purification process. 
2. The purified target protein is contaminant-free, including other proteins, nucleic acids, lipids, and salts.
3. The buffer used in purification is compatible with downstream experiments. 

An effective buffer should exhibit the following properties:

• Water-soluble
• Chemically stable
• pK_a value between 6 and 8
• Minimal effect on the biochemical reactions
• Does not absorb UV light

Chemical components of protein buffers include reagents like Tris or Phosphates or HEPES, salts, detergents, reducing agents, protease inhibitors, imidazole, and protein stabilizers. These constituents help maintain pH, denature or solubilize proteins, minimize damage, and help maximize yield. The chemicals that are used in common extraction buffers may be classified into the following categories:

• Chemicals for pH maintenance
• Salts for ionic balance and stability
• Detergents for denaturing and solubilizing the target protein
• Reducing agents for preventing oxidative damage to the target protein
• Protease inhibitors to inhibit protein degradation
• Co-factors to aid in stabilizing the target protein
• Chelating agents that serve a wide range of purposes

The choice of buffer will be influenced by the type of biological samples to work with (e.g., cells, tissues, microbes). Different biological samples have different cellular structures and compositions, necessitating different buffer formulations. Using the correct buffer for the target sample ensures efficient disruption of cellular membranes and protein solubilization. A buffer that works well for one sample type may not work well for another.

Protein solubility depends primarily on the ionizable amino acid side chains in the polypeptide chain. The solubilization buffer invariably contains buffer substances such as salts and additives that stabilize the proteins. The nature of the buffer used depends on the source of the proteome, the objective of the analysis, and the nature of the workflow to be used afterward.

The concentration and nature of salts also play an important role in regulating protein solubility in an extraction buffer. In general, at low ionic strength, protein solubility increases with increasing salt concentration, but at higher concentrations of salt, proteins tend to precipitate. Protein solubility at a given ionic strength varies with the nature of ions in solution. 

Apart from salts, non-polar compounds such as urea and some detergents are also used to extract and solubilize proteins. Urea also acts as a potent chaotropic agent and brings about the solubilization of proteins. A urea concentration of 8M is sufficient to denature most of the proteins. Several detergents are available and are usually chosen after a pilot screening for a given protein. Detergents can be non-ionic, such as Nonidet® P-40 Substitute, Tween® 20, Tween® 80, Triton® X-100, Triton® X-114, Brij®-35, octyl glucosides etc. Such detergents are usually mild detergents. Detergents can also be categorized as zwitterionic or ionic. CHAPS, amidosulfobetaine-14, and amidosulfobetaine-16 are zwitterionic detergents. Similarly, SDS is an anionic detergent, and CTAB is a cationic detergent. 

Temperature can affect protein denaturation and degradation rates, as well as the efficiency and specificity of the process. Some extractions are performed on ice to preserve protein integrity and minimize unwanted protein modifications and degradation, while others may need to be done at higher temperatures to effectively denature proteins.

The extraction time might vary depending on the sample and proteins of interest, affecting the yield and quality of the extracted proteins. Longer extraction times may be required for tougher tissues or heavily cross-linked samples.

It is important to understand that no single buffer does protein extraction from all the samples and for all the applications, the extraction buffer’s composition needs to be optimized for a particular application, and this requires a screening with available buffers and detergent formulations based on literature survey and a knowledge about the chemistry of constituents.

Finally, referencing established protocols and literature ensures that you are utilizing a buffer that has been shown to work for similar experiments, saving you time and resources.

G-Biosciences provides a range of protein extraction buffers.

FOCUS™ Extraction Buffers

• Chaotropic extraction buffers
• Application: Total protein extraction and solubilization for 2D gel electrophoresis and other applications, rehydration buffer for IPG-strips, improved spot resolution for 2D gel analysis
• Denaturing
• Optimal for proteomic studies

PopLysis™ Protein Extraction Systems

• Optimized kits for working with bacteria, mammalian cells and tissues, neurons, yeast, and insect cells
• Application: Membrane and cytoplasmic protein extraction
• Suitable for downstream applications such as gel electrophoresis, Western blotting, ELISA, chromatography studies, enzyme assays, protein folding studies etc.
• Non-denaturing

Protein Extraction & Lysis Buffer (PE LB™) Systems

• Mild buffer for protein extractions
• Application: release of soluble active proteins from bacteria, yeast, mammalian cells, and mammalian tissues
• Non-denaturing
• Enzyme activity and protein native conformation are preserved
• Suitable for many downstream applications, including enzyme assays, electrophoresis, chromatographic studies, and folding studies 

FOCUS™ Extraction Buffers: https://www.gbiosciences.com/Protein-Research/FOCUS-Extraction-Buffers

PopLysis™ Protein Extraction Systems: https://www.gbiosciences.com/PopLysis_Protein_Extraction_Systems

Protein Extraction & Lysis Buffer (PE LB™) Systems: https://www.gbiosciences.com/Protein-Research/Extraction-and-Lysis-Systems/Protein-Extraction-Lysis-Buffer-PE-LB-Systems

Image 1: Focus extraction buffers 

Protein Purification Handbook