Fonseca et. al. Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydro

Tetra cell system biorad loading volumes were pre

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Unformatted text preview: ysates determined with the bicinchoninic acid (BCA) protein assay (Thermo Scientic, Rockford, IL, USA). Aer electrophoresis, gels were incubated in MMPs– substrate buffer (50 mM Tris–HCl, 10 mM CaCl2, pH 7.5) overnight at 37  C. This buffer is optimal for MMP-2 and MMP-9 gelatine-digestion but also allows detection of other soluble MMPs (including MMP-13).22 Later on, gels were stained with Coomassie Brilliant Blue R-250, followed by several washes in 1 : 2 methanol–acetic acid to reveal MMP proteolytic activity as clear bands against the not-digested gelatine blue background. The same CM (aer APMA activation) was incubated with FRETPVGLIG and FRET-GIVGPL and the enzymatic cleavage of both peptides was estimated by measuring the increase in uorescence aer 48 h. 2.2.3 MMP gene expression (qRT-PCR). RNA was isolated from cells lysed using the TriPure Isolation Reagent (Roche, Mannheim, Germany) and quantied in a NanoDropÒ ND-1000 spectrophotometer using the ND-1000 V3.1.2 soware. For cDNA synthesis, 500 ng of RNA and 1 mL of Random Primers (Invitrogen, Paisley, UK) were diluted 1 : 12 in DEPC-treated H2O, heated to 70  C for 10 min and brought to 4  C. The reverse transcriptase mix (0.7 5 mL DEPC-treated H2O, 4 mL buffer, 1 mL DNTP, 2 mL DTT, 0.2 mL RT, all from Invitrogen Paisley, UK) and 0.2 mL RNasin (Promega, Madison, WI, USA) were added to each sample. Aer reaction (1 h at 37  C) samples were stored at À20  C until further use. For real-time polymerase chain reaction (qRT-PCR) analysis, 0.5 mL cDNA was used per well of a standard PCR 96-well format. Human TaqMan probes were purchased from Applied Biosystems (Foster City, USA): MMP-2 (Hs01548727_m1) and MMP-14 (Hs00237119_m1). qRT-PCR reactions were run on an Applied Biosystems GeneAmp Prism 7700 System and data were analyzed using the 2ÀDct method. Results were normalized to the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Part Number: 4352934E). 2.3 Preparation and characterization of PVGLIG–alginate hydrogel matrices 2.3.1 Synthesis of peptide–alginate bioconjugates. High molecular weight (HMW, Mw ¼ 15 Â 104 Da) sodium alginate was a kind gi from FMC Biopolymers (Protanal LF 20/40, Oslo, Norway). Low molecular weight (LMW, Mw ¼ 3 Â 104 Da) alginate was obtained by g-irradiation of HMW alginate with a cobalt-60 source at a dose of 5 Mrad.23 Peptides were graed to alginate using 1-ethyl-(dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide.12 The oligopeptide GGYGPVGYLIGGK (hereaer designed as PVGLIG, Mw ¼ 1074.24 Da, custom-made by GenScript, Piscataway, NJ, USA) was graed to LMW alginate at a range of 10 to 200 mg of PVGLIG per gram of LMW alginate (herein designated as L10 to L200, respectively). A control sample of LMW alginate that passed through the coupling process but without addition of peptide (L0) was also prepared. The graing efficiency was obtained using the BCA This journal is ª The Royal Society of Chemistry 2013 Soft Matte...
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This document was uploaded on 09/21/2013.

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