CYTOCHROME C PURIFICATION FROM SHEEP HEART VIA ION-EXCHANGE CHROMATOGRAPHY

Cytochrome c purification from sheep heart via ion-exchange chromatography involves several steps to obtain high-purity cytochrome c for further biological or biochemical assays. Here’s the detailed process:

1) Sample Preparation:

• Collect fresh sheep hearts and wash them with phosphate-buffered saline (PBS) to remove any impurities.
• Homogenize the hearts using a blender or sonicator, and centrifuge the homogenate at 10,000 x g for 10 minutes to separate the supernatant and pellet.
• Take the supernatant and dilute it with PBS to a final concentration of 1-2 mg/mL.

2) Ion-Exchange Chromatography:

• Load the diluted supernatant onto an ion-exchange column (e.g., DEAE Sepharose Fast Flow) equilibrated with PBS.
• Wash the column with PBS to remove any impurities and unbound proteins.
• Elute the cytochrome c protein with a gradient of increasing salt concentration (e.g., from 0 to 0.5 M NaCl) in PBS.
• Collect fractions containing cytochrome c based on their absorbance at 420 nm (A420) and protein concentration using a spectrophotometer.

3) Gel Filtration:

• Concentrate the cytochrome c fractions using a centrifugal filter (e.g., Amicon Ultracentrifugal Filter) with a molecular weight cutoff of 30 kDa.
• Load the concentrated cytochrome c onto a gel filtration column (e.g., Sephacryl S-200 HR) equilibrated with PBS.
• Elute the cytochrome c protein with PBS and collect fractions based on their A420 and protein concentration.

4) Final Purification:

• Concentrate the cytochrome c fractions using a centrifugal filter with a molecular weight cutoff of 3 kDa.
• Load the concentrated cytochrome c onto a PD-10 desalting column equilibrated with PBS.
• Elute the cytochrome c protein with PBS and collect the final purified cytochrome c fraction.

The resulting purified cytochrome c should have an A420 of approximately 1.1-1.3 and a protein concentration of 1-2 mg/mL. The purification process can be monitored by SDS-PAGE and Western blot analysis to ensure the presence and purity of cytochrome c.