Journal: Scientific Reports

Article Title: Copper(II)-binding equilibria in human blood

doi: 10.1038/s41598-020-62560-4

Figure Lengend Snippet: Separation of human serum proteins by SEC. 4 x diluted human serum (a), 10 μM Cu•CP ( b ), 10 μM HSA plus 10 μM Cu(II) ( c ), 2.5 μM α2M tetramer plus 10 μM Cu(II) ( d ), 10 μM HSA, 2.5 μM α2M tetramer plus 10 μM Cu(II) ( e ). Conditions: incubation buffer 50 mM HEPES, 50 mM NaCl, pH 7.4; LC-ICP MS: column - 10 × 300 mm Superdex 200; elution buffer 200 mM NH 4 NO 3 , pH 7.4; flow rate 0.4 ml/min; injection volume 10 μl; UV at 280 nm (left axis and black lines) and Cu-63 (right axis and red line), monitored by ICP MS. Figure was created by program Origin 9 Pro ( https://www.originlab.com/ ).

Article Snippet: The experiments were carried out with two preparations of normal (“slow form”) human plasma α2M (from Sigma and Athens Research) as well as with the so-called “fast form” of α2M from Athens Research.

Techniques: Incubation, Injection

Journal: Cancers

Article Title: Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

doi: 10.3390/cancers11030385

Figure Lengend Snippet: Lipocalin 2 (LCN2) expression in blood serum and blood smears withdrawn from HCC patients. ( A ) LCN2 protein secretion in the blood serum of human HCC patients. α-Fetoprotein (AFP) was shown as a known marker elevated in HCC patients, while α 2 -Macroglobulin (α 2 M) was used as a loading control. ( B ) Densitometry of LCN2 results depicted in ( A ). Statistical analysis was performed by Student t-test and standard deviations below 0.05 were considered significant. ( C ) Blood smears were stained for LCN2 by routine immunocytochemistry procedure to visualize LCN2 positive cells. Serum from healthy subjects was used as a control. Magnifications presented are 200× and 400×.

Article Snippet: α2-Macroglobulin , 200-101-207-0100 , poly, goat , Rockland Immunochemicals , h.

Techniques: Expressing, Marker, Control, Staining, Immunocytochemistry

Journal: Cancers

Article Title: Perilipin 5 and Lipocalin 2 Expression in Hepatocellular Carcinoma

doi: 10.3390/cancers11030385

Figure Lengend Snippet: Antibodies used for immunostaining and Western blot analysis.

Article Snippet: α2-Macroglobulin , 200-101-207-0100 , poly, goat , Rockland Immunochemicals , h.

Techniques: Immunostaining, Western Blot

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