Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).
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Method Article
Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
In This Article
Summary
Abstract
"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.
Introduction
"Centromeres" were classically defined as regions of suppressed meiotic recombination in genetics and later recognized as the primary constriction of mitotic chromosomes, which plays an essential role in accurate chromosome segregation during mitosis. "Kinetochores" were described as the multilayered structures that bind to microtubules at the surface of centromeres, as revealed by electron microscopy; "kinetochores" were later defined as the macromolecular complex that localizes at the centromere of mitotic chromosomes. Despite a dramatic divergence of centromeric DNA sequences among vertebrates, kinetochore structure and composition are highl....
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Protocol
1. Cell Culture and Transfection
- Put a cover glass (22 mm x 22 mm) in a 6-well polystyrene plate. Optionally coat a cover glass with Poly-L-Lysine, 0.1% w/v, in water (see List of Materials/Equipment) to keep mitotic cells on the cover glass following the steps below:
Note: Optimal conditions must be determined for each cell line and application.- Aseptically coat culture surface with Poly-L-Lysine, 0.1% w/v, in water (0.4 ml/well of a 6-well polystyrene plate). Rock gently to ensure even coating of the culture surface.
- After 5 min, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water....
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Results
Immunofluorescence analysis of endogenous CENP-A supports the hypothesis that CUL4A-E3 ligase is required for localization of CENP-A to centromeres
Our recent studies showed that CUL4A-RBX1-COPS8 E3 ligase activity is required for ubiquitylation of lysine 124 (K124) on CENP-A and localization of CENP-A to centromeres.17 Initially, the interphase-centromere complex (ICEN) was isolated by anti-CENP-A:native chromatin immunoprecipitation,32-34 and i.......
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Discussion
In recent years many studies have developed different quantitative microscopy assays for fixed cells.42 Progress in centromere-kinetochore biology often requires an understanding of the centromere-specific or kinetochore-specific function of proteins of which subcellular spatial-temporal regulation reflects the changing functions of these proteins during cell cycle. Therefore, here we developed methods of IIF staining and a quantitative IIF assay to specifically analyze the relative levels of endogenous and ex.......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
This work was supported by NIH grant GM68418.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Lipofectamin 2000 | Life Technologies/Invitrogen | 11668 | transfection reagent I |
| Lipofectamin RNAiMAX | Life Technologies/Invitrogen | 13778 | transfection reagent II |
| Opti-MEM I | Life Technologies/Invitrogen | 31985 | Reduced serum media, warm in 37 °C water bath before use |
| High-glucose DMEM (Dulbecco’s modified Eagle’s medium) | Life Technologies/BioWhittaker | 12-604 | high-glucose DMEM, warm in 37 °C water bath before use |
| Fetal Bovine Serum, certified, heat inactivated, US origin | Life Technologies/Gibco | 10082 | FBS (fetal bovine serum) |
| Poly-L-Lysine SOLUTION | SIGMA-SLDRICH | P 8920 | Poly-L-Lysine, 0.1% w/v, in water |
| UltraPure Distilled Water | Life Technologies/Invitrogen/Gibco | 10977 | Sterile tissue culture grade water |
| Micro Cover glass (22 mm x 22 mm) | Surgipath | 105 | Cover glass (22 mm x 22 mm) |
| 6 Well Cell Culture Cluster | Fisher/Corning Incorporated | 07-200-83 | 6-well polystyrene plate |
| Penicillin, Streptomycin; Liquid | Fisher/Gibco | 15-140 | Penicillin-streptomycin |
| PAP PEN | Binding Site | AD100.1 | Hydrophobic barrier pen (for a water repellant barrier in immunofluorescent staining) |
| Paclitaxel (Taxol) | SIGMA-SLDRICH | T7402 | Taxol for mitotic cell analysis |
| TN-16, microtubule inhibitor (TN16) | Enzo Life Sciences | BML-T120 | TN16 for mitotic cell analysis |
| BSA (bovine serum albumin) | SIGMA-SLDRICH | A7906 | Blocking reagent |
| Triton X-100 | SIGMA-SLDRICH | T8787 | Detergent for permeabilization |
| Paraformaldehyde | SIGMA-SLDRICH | P6148 | Fixation reagant |
| DAPI | SIGMA-SLDRICH | D9542 | For nuclear staining |
| p-phenylenediamine | SIGMA-SLDRICH | P6001 | For mounting medium |
| VWR Micro Slides, Frosted | VWR International | 48312-013 | Micro slides |
| Anti-CENP-A antibody | Stressgen/Enzo Life Sciences | KAM-CC006 | Mouse monoclonal antibody; dilution ratio of 1:100 (IIF), 1:5000 (WB) |
| Anti-CENP-B antibody | Novus Biologicals | H00001059-B01P | Mouse monoclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation) |
| Anti-CENP-B antibody | abcam | ab25734 | Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF, methanol/acetone fixation)-1:400 (IIF, paraformaldehyde fixation) |
| Anti-centromere antibody (ACA) | Fitzgerald Industries International, Inc. | 90C-CS1058 | Human centromere antiserum; dilution ratio of 1:2000 (IIF) |
| Anti-CENP-H antibody | Bethyl Laboratories | BL1112 (A400-007A) | Rabbit polyclonal antibody; dilution ratio of 1:200 (IIF) |
| Anti-CENP-H antibody | BD | 612142 | Mouse monoclonal antibody; dilution ratio of 1:200 (IIF) |
| Anti-CENP-I antibody | N/A, Dr. Katusmi Kitagawa | N/A, Dr. Katusmi Kitagawa | Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF); Niikura et al., Oncogene, 4133-4146 (2006) |
| Anti-KNL1 antibody | Novus Biologicals | NBP1-89223 | Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF) |
| Anti-Hec1 antibody | Novus Biologicals / GeneTex | NB 100-338 / GTX70268 | Mouse monoclonal antibody; dilution ratio of 1:100 (IIF) |
| Anti-Hec1 antibody | GeneTex | GTX110735 | Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF) |
| Anti-Ska1 antibody | abcam | ab46826 | Rabbit polyclonal antibody; dilution ratio of 1:100 (IIF) |
| Anti-Flag antibody | SIGMA-ALDRICH | F3165 | Mouse monoclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB) |
| Anti-Flag antibody | SIGMA-ALDRICH | F7425 | Rabbit polyclonal antibody; dilution ratio of 1:1000 (IIF), 1:5000 (WB) |
| Anti-CUL4A antibody | N/A, Dr. Pradip Raychaudhuri | N/A, Dr. Pradip Raychaudhuri | Rabbit polyclonal antibody; dilution ratio of 1:3000 (WB); Shiyanov et al., The Journal of biological chemistry, 35309-35312 (1999) |
| Anti-RBX1 antibody | Cell Signaling | 4397 | Rabbit polyclonal antibody; dilution ratio of 1:2000 (WB) |
| Anti-GAPDH antibody | Chemicon | MAB374 | Mouse monoclonal antibody; dilution ratio of 1:5000 (WB) |
| Alexa Fluor 488 Goat Anti-Mouse IgG | Life Technologies/Invitrogen | A11001 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Alexa Fluor 594 Goat Anti-Mouse IgG | Life Technologies/Invitrogen | A11005 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Alexa Fluor 488 Goat Anti-Rabbit IgG | Life Technologies/Invitrogen | A11008 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Alexa Fluor 594 Goat Anti-Rabbit IgG | Life Technologies/Invitrogen | A11012 | fluorophore-conjugated secondary antibody (Affinity-purified secondary antibody) |
| Non fat powdered milk (approved substitution for carnation powdered milk) | Fisher Scientific | NC9255871 (Reorder No. 190915; Lot# 90629) | Skim milk |
| Leica DM IRE2 motorized fluorescence microscope | Leica | motorized fluorescence microscope | |
| HCX PL APO 63x oil immersion lens | Leica | LEICA HCX PL APO NA 1.40 OIL PH 3 CS | 63X oil immersion lens |
| HCX PL APO 100x oil immersion lens | Leica | LEICA HCX PL APO NA 1.40 OIL PHE | 100X oil immersion lens |
| Leica EL6000 compact light source | Leica | External compact light source for fluorescent excitation | |
| ORCA-R2 Digital CCD camera | Hamamatsu | C10600-10B | digital CCD camera |
| Openlab version 5.5.2 Scientific Imaging Software | Perkin Elmer/Improvision | For image observation, acquisition, quantification, and analysis | |
| Velocity version 6.1.1 3D Image Analysis Software | Perkin Elmer/Improvision | For image observation, acquisition, quantification, and analysis | |
| Complete EDTA-free protease inhibitor cocktail | Roche | 11873580001/11836170001 | Protease inhibitor cocktail tablets |
| PlusOne 2-D Quant Kit | Amersham Biosciences | 80-6483-56 | Commercial protein assay reagent I for measurement of protein concentration (compatible with 2% SDS) |
| Bio-Rad Protein Assay | Bio-Rad | 500-0006 | Commercial protein assay reagent II for measurement of protein concentration (compatible with 0.1% SDS) |
| Immobilon-FL | EMD Millipore | IPFL00010 | PVDF membrane for transferring |
| IRDye 800CW Goat Anti-Mouse IgG | LI-COR Biosciences | 926-32210 | IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF) |
| IRDye 680 Goat Anti-Rabbit IgG | LI-COR Biosciences | 926-32221 | IR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF) |
| Goat anti-Mouse IgG DyLight 549 | Fisher Scientific | PI35507 | DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF) |
| Goat anti-Rabbit DyLight 649 | Fisher Scientific | PI35565 | DyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:20000 (IIF) |
| Goat anti-mouse IgG-HRP | Santa Cruz | SC-2005 | HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF) |
| Goat anti-rabbit IgG-HRP | Santa Cruz | SC-2004 | HRP-conjugated secondary antibodyDyLight-conjugated secondary antibodyIR fluorescent dye-conjugated secondary antibody (Affinity-purified secondary antibody); dilution ratio of 1:10000 (IIF) |
| Openlab version 5.5.2. Scientific Imaging Software | Improvision/PerkinElmer | Software A | |
| Volocity version 6.3 3D Image Analysis Software (Volocity Acquisition) | PerkinElmer | Software B1 | |
| Volocity version 6.3 3D Image Analysis Software (Volocity Quantification) | PerkinElmer | Software B2 | |
| Branson SONIFIER 450 | Sonicator | ||
| Branson Ultrasonics sonicator Microtip Step, Solid, Threaded 9.5 mm | VWR Scientific Products Inc. | 33995-325 | Disruptor horn for sonication |
| Branson Ultrasonics sonicator Microtip Tapered 6.5 mm | VWR Scientific Products Inc. | 33996-185 | Microtip for sonication |
| Odyssey CLx Infrared imaging System | LI-COR Biosciences | Infrared imaging system for immunoblot detection | |
| Image Studio Analysis Software Ver 4.0 | LI-COR Biosciences | Software C | |
| Molecular Imager Versadoc MP4000 System | Bio-Rad | Chemiluminescence imager for immunoblot detection | |
| Quantity One 1-D analysis software | Bio-Rad | Software D | |
| SuperSignal West Femto Maximum Sensitivity Substrate | Thermo | 34095 | Ultra-sensitive enhanced chemiluminescent (ECL) substrate |
References
- DeLuca, J. G., et al. Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites. Molecular biology of the cell. 16, 519-531 (2005).
- Earnshaw, W. C., Halligan, N., Cooke, C., Rothfield, N.
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