Each tumor was sonicated for 30 seconds on ice and centrifuged at 12,000 rpm for 10 minutes. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit (Thermo Scientific, Rockford, IL). Protein levels were calculated from a standard curve obtained with a protein standard, bovine serum albumin (BSA). L-methionine levels were determined with the HPLC procedure described previously [47 (link), 64 (link)]. Standardized L-methionine levels were calculated per mg tumor protein.
Kawaguchi K., Igarashi K., Li S., Han Q., Tan Y., Kiyuna T., Miyake K., Murakami T., Chmielowski B., Nelson S.D., Russell T.A., Dry S.M., Li Y., Unno M., Eilber F.C, & Hoffman R.M. (2017). Combination treatment with recombinant methioninase enables temozolomide to arrest a BRAF V600E melanoma in a patient-derived orthotopic xenograft (PDOX) mouse model. Oncotarget, 8(49), 85516-85525.
Protein levels measured using Coomassie Protein Assay Kit
L-methionine levels determined by HPLC procedure
control variables
Sonication on ice
Centrifugation at 12,000 rpm for 10 minutes
Protein standard (bovine serum albumin, BSA) used for standard curve
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