Quantifying Enolase Activity via Coupled Assay

Enolase activity was measured via NADH oxidation in a pyruvate kinase–lactate dehydrogenase coupled assay as previously described^{12 (link)}. Briefly, cells were lysed in 20 mM Tris HCl, 1 mM EDTA, and 1 mM β-mercaptoethanol (pH 7.4) and homogenized using a Polytron homogenizer three times for a period of 10 s followed by sonication. Enolase activity was recorded by measuring oxidation of NADH either spectrophotometrically by absorbance at 340 nm or fluorescently by excitation at 340 nm and emission at 460 nm.

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Muller F.L., Colla S., Aquilanti E., Manzo V., Genovese G., Lee J., Eisenson D., Narurkar R., Deng P., Nezi L., Lee M., Hu B., Hu J., Sahin E., Ong D., Fletcher-Sananikone E., Ho D., Kwong L., Brennan C., Wang Y.A., Chin L, & DePinho R.A. (2012). Passenger Deletions Generate Therapeutic Vulnerabilities in Cancer. Nature, 488(7411), 337-342.

Publication 2012

Assay Cells Edta Enolase Lactate dehydrogenase Mercaptoethanol Nadh Pyruvate kinase Tris

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, Dana-Farber Cancer Institute, Memorial Sloan Kettering Cancer Center

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Variable analysis

independent variables

Lysis buffer composition (20 mM Tris HCl, 1 mM EDTA, 1 mM β-mercaptoethanol, pH 7.4)
Homogenization method (Polytron homogenizer, three times for 10 s)
Sonication

dependent variables

Enolase activity measured by oxidation of NADH
Oxidation of NADH measured spectrophotometrically (absorbance at 340 nm) or fluorescently (excitation at 340 nm, emission at 460 nm)

control variables

PH of lysis buffer (7.4)

controls

Positive control: Pyruvate kinase–lactate dehydrogenase coupled assay as described in the referenced publication¹²
Negative control: Not explicitly mentioned

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