Rab7 Phosphorylation reaction

Mix phosphorylation reaction mixture. Combine 4 uM human Rab7 with 400 nM purified human strep-tagged TBK1 in a buffer consisting of 50 mM HEPES 7.5, 150 mM NaCl, 10 mM MgCl2, and 200 uM ATP. Incubate at room temperature for 3 hours.

Pass reaction mixture over a strep-tactin sepharose gravity column. Pass reaction mixture over resin x4 in order to remove all strep tagged TBK1 from solution.

Buffer exchange via dilution and centrifugation or dialysis overnight in order to recover purified Rab7

In order to assess degree of phosphorylation, prepare a 15 ug sample of phosphorylated Rab7 for PhosTag gel electrophoresis. Buffer exchange the protein into pure water via dilution and concentration with centrifugal concentrator.

As a positive control, prepare a sample of purified Casein, purchasable in bulk as a lyophilized solid. This protein is natively phosphorylated. Incubate with lambda phosphatase in order to produce its unphosphorylated form. Run the native casein as a positive control for the PhosTag gel, and the unphosphorylated form as a negative control.

Follow guidelines for Fujifilm Wako's "SuperSep" precast PhosTag gel. Add loading buffer to 1x, and run gel at ~120 V until the dye front reaches the bottom of the gel.

Stain gel via Coomassie staining, and image.