sds page gelatin zymography  (Bio-Rad)

Journal: ACS Central Science

Article Title: Redirecting Pore Assembly of Staphylococcal α-Hemolysin by Protein Engineering

doi: 10.1021/acscentsci.8b00910

Figure Lengend Snippet: Cytotoxicity of PAMs toward cultured cancer cell lines, HT-1080 and HL-60 cells. (A) Cytotoxicity of PAMs toward HT-1080 (solid bars) or HL-60 (lined bars) cells. The cytotoxicity was evaluated by using the CytoTox 96 kit (Promega) after 8 h of incubation with PAMαHL or PAMαHLG1; the absorbance at 490 nm representing the concentration of released lactate dehydrogenase was recorded with a microtiter plate reader. Cytotoxicity (%) = (( A s – A spon )/( A max – A spon )) × 100 where A s is the absorbance of toxin-treated cells, A spon is the absorbance of untreated cells (spontaneously dead cells), and A max is the absorbance of cells treated with lysis buffer. (B) Binding assay of PAMs to HT-1080 and HL-60 cells determined by electrophoresis in a 10% SDS-polyacrylamide gel, followed by autoradiography. PAMs (PLHL, PLHLG1, IGHL, IGHLG1; 62 nM) were incubated with HT-1080 cells or HL-60 cells (1 × 10 7 cells mL –1 ) in 5% CO 2 for 40 min at 37 °C. After centrifugation, the resuspended pellets and the supernatants were analyzed. ▲, oligomers; P, pellet; S, supernatant. (C) Extent of MMP-2 expression from HT-1080 and HL-60 cells analyzed by SDS-PAGE gelatin zymography. HT-1080 cells and HL-60 cells were incubated in serum-free DMEM and IMDM, respectively, in 5% CO 2 at 37 °C overnight. The media were collected, centrifuged to remove debris, and mixed with zymogram sample buffer before loading into the zymogram gel (2 μg final concentration of total protein).

Article Snippet: MMP-2 enzymatic activity in tissue culture media was determined by 10% SDS-PAGE gelatin zymography (Ready Gel Zymogram Gel, Bio-Rad).

Techniques: Cell Culture, Incubation, Concentration Assay, Lysis, Binding Assay, Electrophoresis, Autoradiography, Centrifugation, Expressing, SDS Page, Zymography

Journal: PLoS ONE

Article Title: Matrix metalloproteinase promotes elastic fiber degradation in ligamentum flavum degeneration

doi: 10.1371/journal.pone.0200872

Figure Lengend Snippet: A: Representative gelatin zymography analysis of LF fibroblasts in response to IL-6/sIL-6Rα stimulation for 24 h with or without Stattic. B: Quantitative analysis of data shown in panel A (n = 3). C: Concentration of soluble elastin following incubation for 6 h with the supernatant of IL-6-stimulated cultures with or without Stattic (n = 3). Data represent the mean ± SEM. **P < 0.01.

Article Snippet: LF fibroblasts with or without 50 nM Stattic pretreatment were stimulated with IL-6 (300 ng/ml) for 24 h. MMP-2 and -9 activities in the culture supernatant were measured with an SDS-PAGE gelatin zymography kit (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s instructions.

Techniques: Zymography, Concentration Assay, Incubation