I am working on the sulfur metabolism and if I want to isolate a protein from the cytoplasm, which would be the best method to isolate the protein?
Has anyone purified this protein before? Its best to use a protocol that has been used and published. Usually, we make cell lysate and run it on different types of chromatographic colums for separation (Ion exchange, gel fitration, and finally affinity chromatography). You must then check purity of the protein by electrophoresis and activity. Hope this helps!
Well, your question is quite general. What do you know about your protein?
From that information you would be able follow specific techniques, like ultracentrifugation or size-exclusion chromatography (if you know your protein's size), ion exchange chromatography (if you know the isoelectric point), etc.
Chromatographies are the best, but they work much better if you do a previous pre-purification step in order to remove most of the proteins you are not interested in.
For each step you do, remember to save an sample to run it on a SDS gel just to have a visual cue of your purification procedure.
Again, start by asking yourself what do you know about the protein and its properties, it usually will guide you in the right direction.
I purified a cytoplasmic protein by crushing the tissue->centrifuging it down (14,000 x at 4 C for 15-30 mins) -> removing supernatant -> vortexing with equal amount of glass beads for 10 seconds, then incubating on ice for 10 seconds and repeat 5 times -> ultracentrifuging at 100,000 for 1 hr at 4C. Take the pellet, resuspend it then run an SDS-PAGE/western
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