Purification and Characterization of Enzymes

Recombinant toyocamycin nitrile hydratase was expressed and purified as described previously^{24 (link)}. Rabbit pyruvate kinase was purchased from Roche; the phosphoglycerate mutase 2 was discovered as a contaminant in that sample. Glyceraldehyde 3-phosphate dehydrogenase was isolated from HeLa S3 cells as described previously²⁵. Ferritin from horse spleen was purchased from Sigma Aldrich. Finally, human 20S proteasome was purchased from Enzo Life Sciences. All samples were desalted prior to analysis by buffer exchanging with molecular weight cutoff spin filters (Millipore) into 150 mM ammonium acetate at pH 7.

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Skinner O.S., Havugimana P.C., Haverland N.A., Fornelli L., Early B.P., Greer J.B., Fellers R.T., Durbin K.R., Do Vale L.H., Melani R.D., Seckler H.S., Nelp M.T., Belov M.E., Horning S.R., Makarov A.A., LeDuc R.D., Bandarian V., Compton P.D, & Kelleher N.L. (2016). An informatic framework for decoding protein complexes by top-down mass spectrometry. Nature methods, 13(3), 237-240.

Publication 2015

Rabbit pyruvate kinase Ferritin from horse spleen human 20S proteasome molecular weight cutoff spin filters

20s proteasome Ammonium acetate Buffer Ferritin Glyceraldehyde 3 phosphate dehydrogenase Hela cells Horse Human Nitrile hydratase Phosphoglycerate mutase Pyruvate kinase Rabbit Spleen Toyocamycin

Corresponding Organization :

Other organizations : Northwestern University, University of Arizona, Thermo Fisher Scientific (Germany)

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Variable analysis

independent variables

Expression and purification of recombinant toyocamycin nitrile hydratase
Purchasing of rabbit pyruvate kinase
Isolation of glyceraldehyde 3-phosphate dehydrogenase from HeLa S3 cells
Purchasing of ferritin from horse spleen
Purchasing of human 20S proteasome

dependent variables

Measured outcomes of the experiments involving the expressed and purified proteins

control variables

Desalting of all samples by buffer exchanging with molecular weight cutoff spin filters into 150 mM ammonium acetate at pH 7

controls

No positive or negative controls were explicitly mentioned in the provided information.

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