Lithium high performance physiological column

Kidney tissues from mice kidney tissue or from zebrafish embryos, or primary cultured cells were homogenized and lysed with N-ethylmaleimide (NEM) solution containing 5.2 mmol l⁻¹N-ethylmaleide in 10 mmol l⁻¹ potassium phosphate buffer adjusted to pH 7.4. The lysates were collected and precipitated with sulfosalicylic acid (12% w/v) and centrifuged at 10,000 r.p.m. for 10 min at 10 °C. The resulting supernatant was dissolved in citrate loading buffer (Biochrom Ltd, Cambridge, UK) and 50 µl of this solution was analyzed by Biochrom 30 Plus Amino Acid Analyzer (Biochrom Ltd). The protein pellet was dissolved in 0.1 mol l⁻¹ NaOH solution and the protein concentration was determined by Biuret method. The concentration of amino acids was measured by using a lithium high performance physiological column (Biochrom) followed by post-column derivatization with ninhydrin. The amino acids were identified according to the retention time and the ratio of the area between the two wavelengths (570 nm and 440 nm) and quantified by using EZChrom Elite software (Agilent Technologies Inc., Pleasanton, California, USA). Cystine concentration was normalized to the protein concentration and reported in nmol per mg protein. N-ethylmaleimide

Tissue samples from rats or primary cultured cells were homogenized and lysed with N-ethylmaleimide (NEM) solution containing 5.2 mmol l⁻¹N-ethylmaleimide in 10 mmol l⁻¹ potassium phosphate buffer adjusted to pH 7.4. The lysates were collected and precipitated with sulfosalicylic acid (12% w/v) and centrifuged at 10 000 r.p.m. for 10 min at 4°C. The resulting supernatant was dissolved in citrate loading buffer (Biochrom Ltd, Cambridge, UK) and 50 μl of this solution was analyzed by Biochrom 30 Plus Amino Acid Analyzer (Biochrom Ltd). The protein pellet was dissolved in 0.1 mol l⁻¹ NaOH solution and the protein concentration was determined by the Biuret method. The concentration of amino acids was measured by using a lithium high-performance physiological column (Biochrom Ltd) followed by postcolumn derivatization with ninhydrin. The amino acids were identified according to the retention time and the ratio of the area between the two wavelengths (570 and 440 nm) and quantified by using EZChrom Elite software (Agilent Technologies Inc., Pleasanton, CA, USA). Cystine concentration was normalized to protein concentration and reported in nmol per mg protein (11 (link)).