Journal: The American journal of pathology

Article Title: Cystatin C triggers neuronal degeneration in a model of multiple system atrophy.

doi: 10.1016/j.ajpath.2013.11.018

Figure Lengend Snippet: Figure 1 Isolation and identification of candidate signaling molecules to regulate neuronal a-syn accumulation. A: CM samples from primary neural cultures were fractionated by sequential ultrafiltration and monoQ chromatography. The collected proteins were fractionated by Superdex 200 gel filtration chromatography. Protein concentrations in the CM from non-Tg (diamonds) and Tg (circles) mice were estimated by absorbance at 280 nm. B: Fractions 13 to 22 were separated by SDS-PAGE, and the proteins were visualized by silver staining; CM were derived from non-Tg (left panel) and Tg mice (right panel). A 14.9-kDa band (arrow) was specifically detected in fraction 18obtainedfromTgmouseprimarycellsandwasexcisedfromtheSDS-polyacrylamidegelfor massspectrometricanalysis.C:The14.9-kDabandwassubjectedto in-gel digestion with trypsin, and the extracted peptides were analyzed by MALDI-TOF MS. D: Five peptide masses (1256.63, 1412.62, 1542.53, 1811.65, and 1939.65) matched mouse cystatin C. E: The tryptic peptides (bold) cover 42 of 140 amino acids of the cystatin C protein.

Article Snippet: The concentrated fraction in 1.5 mL of CM was supplemented with 20 mmol/L Tris-HCl (pH 7.5) to 10 mL, and the mixture was loaded at 1 mL/minute onto a MonoQ 46/100 PE column (GE Healthcare, Buckinghamshire, UK) preequilibrated with 20 mmol/L Tris-HCl (pH 7.5).

Techniques: Isolation, Chromatography, SDS Page, Silver Staining, Derivative Assay