Akta Pure Protein Purification Protocol
Protein purification is a sequence of processes intended to isolate a singular type of protein from a complex mixture. ÄKTA pure is an adaptable and flexible chromatography system for preparative protein purifications. Here’s a generalized purification protocol using the ÄKTA pure system:
– ÄKTA pure FPLC system
– Appropriate chromatography column
– Buffer Solutions
– Protein sample
– 2 ml Microcentrifuge tubes
1. Prepare Buffers and Protein Sample:
– Create all buffers needed for purification. All buffers should have a pH and ionic strength compatible with the stability and solubility of your target protein.
– Clear the protein sample of any huge, unsoluble materials by centrifugation, ideally at 10,000–20,000 g. Filter your sample for added assurance.
2. System Preparation:
– Switch on the ÄKTA Pure system and log into the software on the designated computer.
– Equilibrate the system by passing enough buffers through it to ensure all air has been removed. This prevents air bubbles, which may disturb the experiments.
3. Column Preparation:
– According to the type of column used, it may need to be equilibrated, sanitized, or cleaned before application of the sample. Follow the instructions provided by the column’s manufacturer.
4. Sample Loading:
– The protein sample is applied to the column in a buffer that will allow it to bind to the chromatography matrix. This can often be a buffer with low ionic strength.
– Bound protein is then eluted off of the column either by applying an elevated concentration of free target molecule in solution (competitive elution) or changing the conditions to those under which the protein is unlikely to bind the ligand (gradient elution).
6. Cleaning and Sanitization:
– After running the samples, run cleaning and sanitization procedures to preserve the column life and ensure reproducible results.
7. Data Analysis:
– After running the samples, analyze the chromatogram produced by the software to identify the fractions where the protein of interest is most likely to be found.
Remember, the protocols can vary hugely because of the diverse nature of proteins and the different types of chromatography that can be used (e.g., ion exchange, Hydrophobic interaction, Gel filtration, and Affinity). You should optimize conditions for the specific protein and column being used. Additionally, always follow the manufacturer’s manual and safety guidelines when operating the ÄKTA purification system.
This is a simplified overview. For a more detailed method, refer to your specific protein’s protocol or contact GE Healthcare Life Sciences’ customer support.