Successful biochemical analysis heavily relies on the effective extraction of biologically active proteins from source materials (e.g., cell and tissue samples). Thus, you need to have an excellent working knowledge of your target protein(s) and use the appropriate extraction buffers for a given experimental design to ensure optimal protein recovery.
Why use a buffer solution?
There are many reasons why you need to use an appropriate buffer solution during the protein extraction process. Here’s how it helps:
- It improves the stability of protein molecules as they are subjected to various forces during lysis and extraction.
- It facilitates the isolation of target proteins from other non-soluble cell components.
- It protects the integrity of the target proteins by keeping them in a stable environment.
What makes a good buffer?
According to Norman Good and colleagues, an effective buffer should exhibit the following properties:
- Water-soluble
- Does not permeate biological membranes
- pKa between 6 and 8
- Has a minimal effect on the biochemical reactions
- Chemically stable
- Does not absorb UV light
- Easy to purify
What are some of the most commonly used buffer solutions?
While there is not one buffer solution that is compatible with all types of proteins, there are some that are applicable for a wide variety of protein types.
Tris-HCl – With an effective pH range of 7.0 to 9.0, this buffer is capable of extracting soluble cytoplasmic proteins. The pH of tris buffers is highly dependent on the temperature and the concentration of the solution.
HEPES-NaOH – HEPES-NaOH is an organic chemical buffer solution that works best between pH 7.2 and 8.2. It is ideally used in cases where enzyme structure and function need to be maintained at low temperatures. However, please take note that HEPES interferes with the Lowry protein assay and is not recommended in systems involving the study of radicals, since it favors the formation of radicals under a wide range of conditions.
Sodium dihydrogen phosphate - disodium hydrogen phosphate – This buffer has a pH range between 5.8 and 8.0 and is usually used when the researcher needs to completely solubilize and denature the target proteins.
NP-40 – This non-ionic buffer solution is widely used for analyzing cytoplasmic or membrane-bound proteins and whole cell extracts. Considering its mild nature, it is also the buffer of choice when you need to retain normal protein functions. Note: If NP-40 fails to extract the target protein from insoluble materials or aggregates, try using RIPA buffer.
RIPA (radioimmunoprecipitation assay buffer) – This buffer is ideally used for whole cell extracts, membrane-bound proteins, and nuclear proteins. The RIPA buffer is compatible with most applications (e.g., protein assays and other protein purification techniques). However, it tends to disrupt weak protein-protein interactions, so take the necessary precautions when working with immunoprecipitation or pull-down assays.
In summation, mild extraction buffers may be required for extracting biologically active proteins for a wide range of downstream applications (e.g., ELISA, gel electrophoresis, Western blotting, chromatographic applications, etc.) while denaturing chaotropic buffers that preserve the native charge of proteins are ideally used for extracting proteins for 2D electrophoresis. In addition, there are several buffers designed to isolate total proteomes from various species (e.g., mammals, plants, insects, bacteria, and yeast) as well as for the solubilization of inclusion bodies.
What are the general considerations when choosing a buffer system?
- Make sure your chosen buffer performs well at the chosen pH. To ensure good results, use one with pKa value within one unit of your desired pH and maintain a concentration between 50 and 100 mM.
- Choose a buffer solution that is chemically stable and does not interfere with the activity of the target protein.
- Consider the buffer’s compatibility with subsequent downstream applications.
