Identification of a human mitotic checkpoint gene: hsMAD2

Science

Volumn 274, Issue 5285, 1996, Pages 246-248

  Li, Yong ^a   Benezra, Robert ^a  

^a MEMORIAL SLOAN KETTERING CANCER CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; NOCODAZOLE; PACLITAXEL; PROTEIN;

ARTICLE; CENTROMERE; CHROMOSOME CONDENSATION; DRUG SENSITIVITY; ELECTROPORATION; GENE EXPRESSION; GENE ISOLATION; GENE LOCATION; HELA CELL; HUMAN; HUMAN CELL; METAPHASE; MITOSIS; MITOSIS INHIBITION; MITOSIS SPINDLE; PRIORITY JOURNAL; TUMOR CELL LINE;

HOMO SAPIENS; SACCHAROMYCES CEREVISIAE;

EID: 10244251532     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5285.246     Document Type: Article

References (31)

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21. 6-tagged hsMAD2 was coupled to CNBr-activated Sepharose 4B (Pharmacia). The polydonal antiserum to hsMAD2 was affinity-purified as described (18). The flow-through of the hsMAD2 affinity column was saved and passed over a protein A-Sepharose column (Pharmacia), and the IgG fraction was eluted to generate anti-hsMAD21 (18). IgG from the corresponding preimmune serum was also isolated. Purified IgGs were extensively dialyzed against phosphate-buffered saline (PBS) and concentrated to 2 mg/ml (anti-hsMAD2 and anti-hsMAD2Δ) or 1.5 mg/ ml (preimmune IgG) in Centricon-30 units (Amicon).
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26. Cells were fixed at -20°C with 100% methanol for 6 min and permeabilized at -20°C with 100% acetone for 30 s. After being blocked in PBS containing 3% bovine serum albumin, cells were stained with affinity-purified anti-hsMAD2 (2 μg/ml) in the blocking buffer for 1 hour. Cells were then washed six times with PBS containing 0.1% Triton X-100 and incubated for 30 min with donkey antibody to rabbit IgG conjugated to fluorescein isothiocyanate (FITC) (1:50 dilution, Amersham), After six washes in PBS, cells were stained with DAPI (0.1 μg/ml in PBS) and mounted. For coimmunostaining of hsMAD2 and centromeres, cells were incubated with both anti-hsMAD2 and human antibody to centromeres (1:100 dilution) for 1 hour, washed as described above, and incubated with donkey antibody to rabbit IgG conjugated to FITC and donkey antibody to human IgG labeled with rhodamine (both at 1:50 dilution, Jackson ImmunoResearch).
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31. We thank R. Davis for the cbf1 null strain, J. Colicelli for the human cDNA library, K. Elkon for the human autoantibody to centromeres, A. Murray for communicating unpublished results, and J Roberts for critical reading of the manuscript. Y.L. is a Jack and Susan Rudin Scholar in the Biomedical Sciences. Supported by an NSF grant (IBN-9118977) to R.B. and a core National Cancer Institute grant (P30-CA-08748).