Immunoblotting Protocol for Centromeric Proteins

Cells were suspended in lysis buffer (50 mM Na Phosphate pH 8.0, 30 mM NaCl, 0.1% NP40, 5 mM β-mercaptoethanol, protease inhibitors (Roche) and phosphatase inhibitors) and then resuspended in 2 × SDS loading buffer. The following antibodies were used for immunoblotting: anti-GFP (1:2,500, MBL international), anti-Hec1(1:1,500, Abcam), anti-tubulin (1:4,000, Sigma), anti-CENP-T (1:5,000), anti-CENP-C (1:5,000), horseradish peroxidase-conjugated anti-rabbit IgG (1:100,000, Jackson ImmunoResearch) and horseradish peroxidase-conjugated anti-mouse IgG (1:100,000, Jackson ImmunoResearch)13 (link)35 (link).

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Suzuki A., Badger B.L, & Salmon E.D. (2015). A quantitative description of Ndc80 complex linkage to human kinetochores. Nature Communications, 6, 8161.

Publication 2015

protease inhibitors anti-GFP anti-Hec1 anti-tubulin horseradish peroxidase-conjugated anti-rabbit IgG horseradish peroxidase-conjugated anti-mouse IgG

Anti igg Anti t Antibodies Buffer Cells Cenp c Hec1 Horseradish peroxidase Inhibitors Mercaptoethanol Mouse Nacl Phosphatase Phosphate Protease inhibitors Rabbit Tubulin

Corresponding Organization : University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Lysis buffer composition (50 mM Na Phosphate pH 8.0, 30 mM NaCl, 0.1% NP40, 5 mM β-mercaptoethanol, protease inhibitors, and phosphatase inhibitors)

dependent variables

Protein expression levels of GFP, Hec1, tubulin, CENP-T, and CENP-C

control variables

Unspecified

controls

Positive controls: None specified
Negative controls: None specified

Annotations

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