How to operate the protein purification workstation

Protein purification workstation operating methods often involve multiple meticulous and critical steps to ensure efficient isolation of target proteins from complex mixtures. The following is a general operation method for reference only. Specific operation details may vary depending on equipment models, purification methods, and experimental requirements.

1. Preparation

1. Sample preparation:

1>Collect and process samples containing target proteins, such as cell lysates, tissue extracts or fermentation broth, etc.

2>Depending on the characteristics of the target protein, pretreatment may be required, such as removing nucleic acids, adjusting pH, removing impurities, etc.

2. Equipment inspection:

1> Ensure that all components of the protein purification workstation are intact, connected correctly, and the power supply is stable.

2> Check whether the purification media (such as chromatography columns, affinity chromatography packing, etc.) have been installed correctly and are in working condition.

3. Buffer preparation:

1> According to experimental needs, prepare buffers of different concentrations, including equilibrium buffer, elution buffer, etc.

2> Ensure that the buffer composition, pH value and ionic strength match the properties of the target protein.

2. Sample loading

1. Sample filtering:

1>Use an appropriate filter (such as 0.22μm or 0.45μm filter membrane) to remove impurities and particles in the sample to ensure that the sample is clear and free of suspended solids.

2. Sample loading operation:

1>Add the processed sample into the purification workstation through the sample loading system.

2>Adjust parameters such as flow rate, pressure, and sample loading according to equipment requirements.

3. Purification process

1. Balance:

1>Rinse the purification medium with equilibration buffer to remove impurities and residues in the medium and allow the medium to reach a stable state.

2> Pay attention to the changes in flow rate and pressure during the balancing process to ensure stable operation of the system.

2. Adsorption:

1>After equilibrium, the target protein will bind to the ligands (such as antibodies, metal ions, etc.) on the purification medium to form a complex.

2>Select appropriate adsorption conditions (such as temperature, pH value, ionic strength, etc.) based on the binding characteristics of the target protein and ligand.

3. Elution:

1> By gradually increasing the concentration of the elution buffer or changing other conditions (such as pH value, ionic strength, etc.), the target protein is separated from the purification medium and flows out with the eluent.

2> During the elution process, you should pay close attention to the changes in the elution curve so that you can adjust the elution conditions in a timely manner.

4. Collection and testing

1. Collection:

1>Use a collector to collect the target protein in the eluate.

2>According to experimental needs, the collected liquid may need to be further processed, such as concentration, liquid replacement, etc.

2. Detection:

1>Use appropriate methods to detect the purity and concentration of the target protein. Commonly used methods include SDS-PAGE gel electrophoresis, Coomassie brilliant blue staining, UV spectrophotometry, etc.

2>Record and analyze the result data to evaluate the purification effect.

5. Follow-up processing

1. Data processing:

1>Analyze the purification effect based on the test results, and adjust the experimental conditions to optimize the purification process.

2>Compile experimental data and write experimental reports.

2. Equipment cleaning and maintenance:

1>After purification, all parts of the purification workstation should be cleaned in time to remove residues and dirt.

2> Carry out regular maintenance and upkeep on the equipment to ensure its long-term stable operation.

6. Precautions

1. Operating procedures and safety regulations should be strictly followed during the entire purification process to ensure the safety of experimental personnel and equipment.

2. The selection of purification media and the setting of purification conditions should be rationally designed based on the properties of the target protein and experimental requirements.

3. Pay attention to observe and record the changes in various parameters during the experiment so that problems can be discovered and solved in time.

Since there may be operational differences among different brands and models of protein purification workstations, you should refer to the operating manual of the specific equipment or contact the equipment supplier for more detailed operating instructions during actual operations.