JoVE Encyclopedia of Experiments
Microbiology
0 views • 4:28 min • October 30th, 2025
Take a culture of Streptococcus mutans expressing a polyhistidine-tagged protein secreted into the medium.
Centrifuge the culture and collect the protein-rich supernatant.
Add ammonium sulfate and incubate with stirring to precipitate proteins by reducing their solubility.
Transfer the solution to a tube, centrifuge, and discard the supernatant.
Resuspend the pellet in binding buffer containing salts and low imidazole to stabilize the protein.
Dialyze the solution against fresh binding buffer to remove excess salts.
Centrifuge the dialyzed solution to pellet insoluble debris, then filter the supernatant.
Mix the filtrate with nickel-charged affinity resin and incubate to allow selective binding of the polyhistidine-tagged protein to the nickel ions.
Load the mixture onto the chromatography column and wash with binding buffer to remove unbound proteins.
Next, add an elution buffer with high imidazole to displace histidine from nickel ions and release the polyhistidine-tagged protein.
Collect this eluate containing the purified proteins.
Centrifuge the bacterial culture suspension for 20 minutes at 10,000 times g at four degrees Celsius.
Transfer the culture supernatant into a three-liter glass
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