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4354
Phospho-PLK1 (Thr210) Antibody (ELISA-Specific)
Primary Antibodies
Polyclonal Antibody
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Phospho-PLK1 (Thr210) Antibody (ELISA-Specific) #4354

Citations (3)
Validation of Phospho-PLK1 (Thr210) Antibody (ELISA-Specific) in peptide DELFIA® assay using phospho-, nonphospho-peptide controls, and DELFIA® secondary antibodies (available from Perkin Elmer Life and Analytical Sciences). At 1ug/ml the S/N=84 for Peptide 1 (VEYDGERKKT*L) , while the S/N=134 for Peptide 2 (GERKKT*LCGTPNYI), (n=2).
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Supporting Data

REACTIVITY H
SENSITIVITY
MW (kDa) N/A.
SOURCE Rabbit

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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ELISA Peptide

A. Solutions and Reagents

  1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3 (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer.
  2. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
  3. Wash Buffer: 1X PBS containing 0.05% Tween-20 (PBST)
  4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
  5. Antibody Dilution Buffer: 3% BSA in PBST
  6. DELFIA® Europium-labeled Anti-mouse IgG for mouse primary antibodies or Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124) for rabbit primary antibodies.
  7. DELFIA® Enhancement Solution (PerkinElmer Life Sciences #1244-105)

(DELFIA® is a registered trademark of PerkinElmer, Inc.)

B. Protocol

  1. Coat the wells of a 96-well microtiter plate with 100 μl of 1 μM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hrs at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
  2. Wash plate three times 200 μl/well with wash buffer.
  3. Block plate with 200 μl/well blocking buffer for 1 hr at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)
  4. Prepare appropriate dilution of primary antibody with antibody dilution buffer. Add 100 μl to wells and incubate at 37°C for 1 hr.
  5. Wash three times with wash buffer.
  6. Add 67 ng/well DELFIA Europium-labeled Anti-mouse IgG or Anti-rabbit IgG, diluted in 100 μl/well antibody dilution buffer. Incubate at room temperature for 30 min, on gentle shaker.
  7. Wash three times with wash buffer.
  8. Add 100 μl enhancement solution and incubate at room temperature for 5 min. Read plate at 615 nm with an appropriate time-resolved plate reader.

posted June 2005

revised September 2007

Protocol Id: 34

Specificity / Sensitivity

Phospho-PLK1 (Thr210) Antibody (ELISA-Specific) is phospho-specific by ELISA, but detects multiple bands by Western blot.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Mouse, Xenopus, Pig

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr210 of human PLK1. Antibodies are purified by protein A and peptide affinity chromatography.

Background

At least four distinct polo-like kinases exist in mammalian cells: PLK1, PLK2, PLK3, and PLK4/SAK (1). PLK1 apparently plays many roles during mitosis, particularly in regulating mitotic entry and exit. The mitosis promoting factor (MPF), cdc2/cyclin B1, is activated by dephosphorylation of cdc2 (Thr14/Tyr15) by cdc25C. PLK1 phosphorylates cdc25C at Ser198 and cyclin B1 at Ser133, causing translocation of these proteins from the cytoplasm to the nucleus (2-5). PLK1 phosphorylation of Myt1 at Ser426 and Thr495 has been proposed to inactivate Myt1, one of the kinases known to phosphorylate cdc2 at Thr14/Tyr15 (6). Polo-like kinases also phosphorylate the cohesin subunit SCC1, causing cohesin displacement from chromosome arms that allow for proper cohesin localization to centromeres (7). Mitotic exit requires activation of the anaphase promoting complex (APC) (8), a ubiquitin ligase responsible for removal of cohesin at centromeres, and degradation of securin, cyclin A, cyclin B1, Aurora A, and cdc20 (9). PLK1 phosphorylation of the APC subunits Apc1, cdc16, and cdc27 has been demonstrated in vitro and has been proposed as a mechanism by which mitotic exit is regulated (10,11).

Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). In addition, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis; DNA damage prevents phosphorylation at these sites (14).

Substitution of Thr210 with Asp has been reported to elevate PLK1 kinase activity and delay/arrest cells in mitosis, while a Ser137Asp substitution leads to S-phase arrest (12). Additionally, while DNA damage has been found to inhibit PLK1 kinase activity, the Thr210Asp mutant is resistant to this inhibition (13). PLK1 has been reported to be phosphorylated in vivo at Ser137 and Thr210 in mitosis, and DNA damage prevents phosphorylation at these sites (14).

  1. Nigg, E.A. (1998) Curr Opin Cell Biol 10, 776-83.
  2. Toyoshima-Morimoto, F. et al. (2002) EMBO Rep 3, 341-8.
  3. Toyoshima-Morimoto, F. et al. (2001) Nature 410, 215-20.
  4. Peter, M. et al. (2002) EMBO Rep 3, 551-6.
  5. Jackman, M. et al. (2003) Nat Cell Biol 5, 143-8.
  6. Nakajima, H. et al. (2003) J Biol Chem 278, 25277-80.
  7. Sumara, I. et al. (2002) Mol Cell 9, 515-25.
  8. Hauf, S. et al. (2001) Science 293, 1320-3.
  9. Peters, J.M. (1999) Exp. Cell Res. 248, 339-49.
  10. Kraft, C. et al. (2003) EMBO J 22, 6598-609.
  11. Kotani, S. et al. (1998) Mol Cell 1, 371-80.
  12. Jang, Y.J. et al. (2002) J Biol Chem 277, 44115-20.
  13. Smits, V.A. et al. (2000) Nat Cell Biol 2, 672-6.
  14. Tsvetkov, L. and Stern, D.F. (2005) Cell Cycle 4, 166-71.
  15. Jang, Y.J. et al. (2002) J Biol Chem 277, 44115-20.
  16. Smits, V.A. et al. (2000) Nat Cell Biol 2, 672-6.
  17. Tsvetkov, L. and Stern, D.F. (2005) Cell Cycle 4, 166-71.

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