Journal: The Journal of Biological Chemistry

Article Title: Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca 2+

doi: 10.1074/jbc.M112.364729

Figure Lengend Snippet: Complex formation with α2-macroglobulin and inhibition of MMP-1 enzymatic activity. A, 1 μg of activated MMP-1 was incubated with 1 μl of Zucker rat serum in a final volume of 30 μl and incubated for 30 min at room temperature. Gel sample buffer was added to the completed reaction, and the sample was analyzed by 4–20% SDS-PAGE. After transfer to a nitrocellulose membrane, Western blot analysis was performed with an anti-MMP-1 antibody. 4 μg of total protein from perfusate samples of Zucker rat skin treated with 50 μg/ml MMP-1 (lane 4) and 50 μg/ml GVSK (lane 5) was also analyzed along with Zucker rat serum (lane1), purified MMP-1 (lane 2), and Zucker rat serum + purified MMP-1 (lane 3). Molecular masses (MW) are indicated to the left of the blot in kilodaltons. B, 12 μg of total perfusate protein from rat skin treated with 50 μg/ml MMP-1 was incubated with an anti-α2-macroglobulin antibody. After overnight incubation, protein A/G beads were added and further incubated. At the end of the incubation, gel sample buffer was added, and the samples were analyzed by 4–20% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an anti-MMP-1 antibody. In addition, 1 μg of purified α2-macroglobulin and activated MMP-1 and perfusate from buffer treatment alone served as controls. Molecular masses (MW) are indicated to the left of the blot in kilodaltons. C, MMP-1 and GVSK at 1 μg/ml were incubated in 10% Zucker rat serum containing either 1 mm or 10 mm Ca2+ in a final volume of 120 μl at 25 °C for the indicated times. After incubation the fluorogenic peptide substrate was added to the mixture, and the enzymatic activity was determined. The activity at each time point is plotted as a percentage of the initial specific activity (t = 0). Error bars indicate S.D.

Article Snippet: For the immunoprecipitation experiments, ∼12 μg of total protein from the rat perfusates (∼15–30 μl) was mixed with 5 μl of an anti-rat α2-macroglobulin rabbit antibody (Alpha Diagnostic International) and brought to a final volume of 200 μl with PBST.

Techniques: Inhibition, Activity Assay, Incubation, SDS Page, Western Blot, Purification

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Antibodies, lectins and proteins, as well as their molecular weights, transfer time and incubate time.

Article Snippet: Rabbit anti-α2 macroglobulin (A2M) antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Molecular Weight, High Molecular Weight

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Comparison of the binding ability of PVDF membrane and NC membrane to high molecular weight proteins. ( a ) The mixed sera of healthy samples were diluted by gradient (3.0, 1.5, 0.8, 0.4, 0.2 and 0.1 μg). Then the sera were separated by 8% SDS-PAGE. The proteins were transferred onto PVDF membranes (up) and NC membrane (down), respectively. The membranes were incubated with anti-CerP, anti-IgG and anti-A2M. ( b ) Staining intensities were statistically analyzed (n = 3 individual experiments). Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. *Significantly different p < 0.05, ** p < 0.01, *** p < 0.001. All values are means ± S.E. (error bars).

Article Snippet: Rabbit anti-α2 macroglobulin (A2M) antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Comparison, Binding Assay, Membrane, High Molecular Weight, SDS Page, Incubation, Staining, Software

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Comparison of the re-probed ability of PVDF membrane and NC membrane. The pooled sera proteins (3.0, 1.5, 0.7, 0.3 and 0.1 μg) were separated by 8% SDS-PAGE, and transferred to PVDF membranes (up) and NC membrane (down), respectively. ( a ) Staining with AAL and then re-probed with PHA-E; ( b ) staining with ApoA1 and then re-probing with IgG; ( c ) Staining with PHA-E and then re-probing with A2M; ( d ) staining with A2M and then re-probing with PHA-E. Band intensities were statistically analyzed (n = 3 individual experiments) and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed *Significantly different p < 0.05, ** p < 0.01. N.S., not significant. All values are means ± S.E. (error bars).

Article Snippet: Rabbit anti-α2 macroglobulin (A2M) antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Comparison, Membrane, SDS Page, Staining, Software