During cell lysis, the extracted proteins can become denatured or damaged. They can also separate from the assay solution, or be contaminated by exposure to lipids, DNA, and other irrelevant cell components. To avoid this from happening, researchers use a buffer solution, which helps ensure the integrity and stability of the proteins. Below, we look at some key factors to consider in designing a suitable protein purification buffer.
- Buffer System
Buffer systems are designed to resist change in the assay solution’s pH. These systems have significant amounts of weak acid and its conjugate base (formed when the acid donates a proton), or a weak base and its conjugate acid added to the assay solution. The goal is to create a buffer that has a pKa value within a single pH unit of the optimal pH.
- pH Level
Speaking of pH: researchers need to identify the suitable pH level for the protein of interest. Many researchers aim for pH 7.4, because this considered the healthiest pH level for blood (specifically, between 7.35pH – 7.45pH), and therefore aligns with ideal biological conditions.
- Additives & Agents
Researchers also need to add various additives or agents to the buffer in order to enhance protein solubility and stability. Examples include bovine serum albumin (a.k.a. BSA or Fraction V), which derives from cows, or small amounts of citrate or detergents. Researchers may also need to add viscosity, which can be done by adding an agent like polyethylene glycol (a polyether compound).
- Salt
Researchers often use salt to both enhance protein solubility and stability, as well as align with ideal or desired biological conditions. Often, the salt concentration must be modified (via dialysis of the protein in a new buffer) while protein purification is taking place, in order to avoid nonspecific interactions, and to detect ionic interactions.
- Reducing Agents
If oxidation is a risk factor, then researchers may need to add reducing agents to their protein purification bugger, such as DTT, TCEP, 2-mercaptoethanol etc. Many researchers prefer TCEP because it acts in a broader range of pH, and because it’s very stable. However, it is quite costly, which makes it prohibitive in some research programs.
The Bottom-Line: It’s All About Yield!
When it comes to cell lysis and examining isolated proteins, the objective that all life science researchers have – and especially laboratory managers -- is the same, regardless of whether they work in the food industry, biotech, pharmaceutical field, or anywhere else: it’s all about yield!
At Pion, all of our laboratory homogenizers are built for reliability, and designed to produce repeatable and scalable results, so that researchers can maximize yield in the fewest passes possible – and ultimately scale up to manufacturing or clinical trial as rapidly, reliably and cost-effectively as possible. Learn more here.
Now that you've decided on a buffer, which cell lysis method are you going to use?
