Assay Method Information

Assay Name:  Inhbition Assay
Description:  For expression of hPDHK2 activity, Escherichia coli strain BL21(DE3) cells (Novagen) were transformed with the pET17b vector containing modified hPDHK2 cDNA. The Escherichia coli were grown to an optical density 0.6 (600 nmol/L) at 30 C. Protein expression was induced by the addition of 500 umol/L isopropyl-beta -thiogalactopyranoside. The Escherichia coli were cultured at 30 C. for 5 hr and harvested by centrifugation. Resuspension of the Escherichia coli paste was disrupted by a microfluidizer. FLAG-Tagged protein was purified using FLAG affinity gel. The gel was washed with 20 mmol/L HEPES-NaOH, 500 mmol/L sodium chloride, 1% ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0), and the binding protein was eluted with 20 mmol/L HEPES-NaOH, 100 ug/mL FLAG peptide, 500 mmol/L sodium chloride, 1% ethylene glycol, and 0.1% Pluronic F-68 (pH 8.0). The eluted fractions containing FLAG-Tagged protein were pooled, dialyzed against 20 mmol/L HEPES-NaOH, 150 mmol/L sodium chloride.
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail