Why was it so important to keep all cellular fractions at or near 0 degree C throughout the entire isolation/purification procedure?
To understand why it is important to keep cellular fractions at or near 0 degrees Celsius throughout the isolation/purification procedure, we need to consider the effect of temperature on cellular components and processes.
The primary reason for maintaining a low temperature is to slow down or inhibit enzymatic activity. Enzymes are proteins that catalyze various biochemical reactions within cells. By reducing the temperature, the kinetic energy of the molecules decreases, thereby slowing down the activity and reducing the chance of enzyme-mediated degradation or modification of cellular components.
Furthermore, low temperatures preserve the structural integrity of cellular organelles and biomolecules. Proteins, lipids, and nucleic acids are vulnerable to denaturation, degradation, or aggregation at higher temperatures. Cooling the samples minimizes these risks, ensuring that the cellular fractions remain intact and stable throughout the procedure.
To achieve and maintain a temperature near 0 degrees Celsius during isolation/purification, the following steps can be taken:
1. Pre-cooling: Prior to starting the procedure, ensure that all equipment, buffers, and reagents are chilled in a cold room or a refrigerator at 4 degrees Celsius or lower for a sufficient amount of time. This allows for the transfer of heat from the samples to the surrounding environment.
2. Use ice baths or cold plates: During the isolation and purification steps, keep the sample tubes or containers on ice baths or cold plates to maintain the desired low temperature. These provide a constant source of cooling and help offset any external temperature increases.
3. Minimize exposure to room temperature: Whenever possible, handle the samples in a cold room or on an ice-based surface to prevent them from warming up. Frequent transitions between the cold environment and room temperature should be minimized to maintain the low temperature consistently.
4. Work quickly: The entire procedure should be carried out expeditiously to minimize the exposure time of cellular fractions to room temperature. This reduces the chances of any temperature-induced damage or alterations to the cellular components.
By implementing these precautions and maintaining low temperatures, the integrity and functionality of cellular fractions can be preserved during isolation and purification processes.