HHMI investigator Arul Chinnaiyan and colleagues at the University of Michigan showed that as prostate cancer develops and progresses, sarcosine
levels increase in both tumour cells and urine samples, suggesting that measurements of the metabolite could aid in non-invasively diagnosing the disease.
The agarose plug was incubated for 24 h at 37[degrees]C in 500 [micro]l of lysis buffer (6 mM Tris-HCl, pH 7.6, 0.1 M EDTA, 1 M NaCl, 0.5 per cent Brij 58, 0.4 per cent sodium deoxycholate, 0.5 per cent sodium lauryl sarcosine
(sarcosyl), lysozyme, 1 mg/ml).
Briefly, equal amounts of bacterial suspension and 2% low melting agarose (Sea Plaque, FMC Bioproducts, Rockland, ME) were pipetted into plug molds and incubated in ESP buffer (0.5 M EDTA, 1% N-lauroyl sarcosine
, 1 mg/mL Proteinase K; pH 8.5-9.3) overnight at 50[degrees]C.
An equal volume of lysis buffer (0.2 M Tris, 0.05 M EDTA, 2.0 M NaCl, 2% hexadecyltrimethylammonium bromide) was added to the tube, followed by 5% sarcosine
. The tubes were incubated at 65 [degrees] C to completely lyse the cells.
Then was add an equal volume of EC solution (Tris-HCl pH 7.6, 6 mM; NaCl 1M; EDTA pH 7.5 1 mM; Brij-58 0.5%; sodium deoxycholate 0.2%; lauril sarcosine
0.5%) and was incubated overnight at 37[degrees]C, the solution was eliminated and then was add an equal volume of ESP solution (EDTA pH 9.5, 0.5M; lauril sarcosine
, 1%; proteinase K, 1 mg/mL), with gently shake, incubated by two days at 50[degrees]C.
Koo, "Implications of differences in expression of sarcosine
metabolism-related proteins according to the molecular subtype of breast cancer," Journal of Translational Medicine, vol.
The greatest degree of imprecision was associated with those amino acids present at concentrations <10 [micro]mol/L including homocitrulline, sarcosine
, and [gamma]-aminobutyrate.
Plasma creatinine and urea nitrogen were determined using commercial kits (sarcosine
oxidase-peroxidase-antiperoxidase; Zixing, Shanghai, China).
Lysis solution (2.5 M NaCl 100 mM EDTA 10 mM Tris (pH 10) 1% N-lauroyl sarcosine
sodium 1% Triton X-100 and 10% DMSO) and alkaline buffer (1 mM EDTA 300 mM NaOH) were prepared in the lab.
Based on the results of tests, the aqueous potassium salts (composed of sarcosine
and proline) are considered to be the most promising solvents.
During this reaction, dimethylglycine is degraded to sarcosine
and finally to glycine.
After induction by isopropyl[beta]-D-thiogalactopyranoside, the cells were sonicated, and the recombinant proteins were extracted with 1.5% sarcosine
. Finally these recombinant proteins were bound by BD TALON metal affinity resins (BD Biosciences, San Jose, CA) and examined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.