I am trying to purify a plasma membrane protein from ecoli.
I think the standard procedure for membrane protein extraction is like this:
However, sometimes proteins are in the pellet after the low speed spin (step 2). I think this is referred to as insoluble fraction typically.
For proteins in this pellet, does this guarantee the protein is in inclusion bodies?
Or can properly folded, hydrophobic plasma membrane proteins naturally be here without inclusion bodies?
Normally, the precipitation components obtained by low-speed centrifugation include cell nuclei and a few unbroken cells, and then the precipitate obtained by high-speed centrifugation with supernatant is the cell membrane.
At 5000 xg, you are pelleting mainly unbroken cells, and some inclusion bodies, so you may have a mix of properly folded and misfolded protein.
Membrane protein and Inclusion body have their own physical characteristics.
Suppose your target protein expression is huge and formed inclusion body , you will get completely white, rubbery, solid pellet. It will not dissolve easily by detergent treatment. If you vortex vigorously, it will form a turbid colloidal solution.
On the other hand, protein expressed in the membrane, formed a fluffy and slimy layer on the insoluble part after low speed spin centrifugation (surface of the pellet). This will easily get soluble by mild vortexing in detergent containing buffer.
Each step you can analyze by SDS-PAGE gel and have an idea between membrane protein and inclusion body.
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