In gel tryptic digestion kit

After HAC-2D MICS-BN-PAGE, silver or CBB staining was conducted for the slab gel to detect the spots of isolated metalloprotein that had migrated off the diagonal line. The spots cut from the resulting slab gel were then subjected to in-gel tryptic digestion using the In-Gel Tryptic Digestion Kit (Thermo Fisher Scientific, Waltham, USA), according to the kit protocol. Digested sample solutions were desalted and concentrated by GL-TipTM SDB (GL Sciences, Tokyo, Japan) prior to being subjected to MALDI-TOF MS. For MALDI-TOF MS measurements, α-cyano-4-hydroxycinnamic acid (HCCA, Wako; for proteome) was employed as the matrix. As a calibration standard, a Peptide Calibration Standard II solution (Bruker Daltonics, Billerica, USA) was employed. The MALDI-TOF MS measurements by Autoflex III (Bruker Daltonics) were conducted using the reflector positive method (with molecular weight range 1–5 kDa). The protein was identified by peptide fingerprinting using publicly available databases (MASCOT, MatixScience).

Purified scintillon preparations were concentrated by ultra-centrifugation at 4°C. The pellet was re-suspended in SDS sample buffer, heated for 5 minutes at 95°C, and cleared by centrifugation at 10,000 × g. 50 μl of sample was loaded and run on NovexNuPAGE 4–12% bis-tris gels according to the manufacturer’s protocol. Gel bands corresponding to the location of the presumptive proteins were excised with a clean scalpel. Samples were processed using the Thermo Scientific In-Gel Tryptic Digestion Kit according to manufacturer’s protocol. Gel bands were destained twice with 200 μl destaining solution (~25 mM sodium bicarbonate in 50% acetonitrile) and incubated at 37°C with shaking for 30 minutes. Samples were reduced by incubation at 60°C for 10 min in 50mM TCEP (tris(2-carboxyethyl)phosphine) in 25 mM ammonium bicarbonate buffer. Free sulfhydryl groups were alkylated by incubation in 100 mM iodoacetamide at room temperature for 1 hour in the dark. Gel pieces were shrunk in acetonitrile. For the initial proteomics run samples were treated overnight with 100 ng trypsin at 30°C. The second proteomics run samples were treated with 100ng typsin and digestion was performed at 50°C at high pressure using the PBI Barocyler (Pressure Biocsciences Inc.) according to manufacturer’s protocol. Samples were dried in a SpeedVac.

HCEC cells were stimulated for 72 h with DAC (10 µM) or vehicle with/without the addition of TNFα (20 ng/mL) for the last 24 h. Proteins of whole-cell lysates were separated by SDS-PAGE. After incubation with a colloidal Coomassie staining solution, bands found at 50 kDa were excised, and an In-Gel Tryptic Digestion Kit (Thermo Scientific^TM PIERCE^TM) was applied. The resulting digest was mixed 1:1 with the matrix solution (α-cyano-4-hydroxy cinnamic acid, HCCA) and 1 µl of this mixture was applied onto a steel plate for MALDI-TOF-MS analysis (Bruker Daltonic GmbH, Bremen, Germany). TOF-MS measurements were performed in the reflector mode operation over the mass range of m/z 500–5000. Further details for methodical parameters are given in the here cited literature [79 (link)].