Isolation and Purification of Recombinant Myelin Oligodendrocyte Glycoproteins

MOGtag is a recombinant Myelin Oligodendrocyte Glycoprotein fused with solubilization and purification tags for Experimental Autoimmune Encephalomyelitis studies.

To isolate MOGtag, suspend bacterial cells expressing MOGtag in a lysis buffer and incubate.

Enzymes and detergents in the buffer disrupt membrane lipids and degrade the cell wall.

Sonicate to lyse the cells further, releasing insoluble inclusion bodies containing MOGtag.

Centrifuge to pellet the inclusion bodies. Discard the supernatant, resuspend the pellet in buffer, and incubate.

Add guanidine hydrochloride, then incubate to solubilize MOGtag.

Transfer the soluble proteins onto a charged nickel resin, mix, and incubate with shaking to allow MOGtag to bind to the nickel ions.

Centrifuge to pellet the bound MOGtag. Remove the supernatant, resuspend the pellet in an elution buffer, and incubate with shaking.

Imidazole in the elution buffer releases MOGtag from the resin.

Centrifuge again, collect the purified MOGtag-containing supernatant, and store it for further analysis.

Distribute the cultures evenly amongst 250 milliliter bottles compatible with high speed centrifugation, and keep the bottles on ice from this point forward.