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Product Pathways - DNA Damage

Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb #54762

Item# Description List Price Web Price Qty
54762S Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb - 100 µl $433.00
$389.70
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VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
Application Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W Human Endogenous 32 Rabbit IgG
IF-IC
F

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry

Protocols

Specificity / Sensitivity

Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb recognizes endogenous levels of RPA32/RPA2 protein only when phosphorylated at Ser8.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser8 of human RPA32/RPA2 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and 293 cells, untreated or treated with UV (100 mJ/cm2, 2 hr recovery), using Phospho-RPA32 (Ser8) (E5A2F) Rabbit mAb (upper) or total RPA32/RPA2 Antibody #52448 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with UV (100 mJ/cm2, 2 hr recovery; right), using Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 594 Phalloidin #12877 (red). Blue = DAPI #4083 (fluorescent DNA dye).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of 293T cells, untreated (blue) or treated with UV (100 mJ/cm2, 4 hr recovery; green), using Phospho-RPA32 (Ser 8) (E5A2F) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.


Background

RPA70 (HSSB, REPA1, RF-A, RP-A, p70) is a component of a heterotrimeric complex, composed of 70, 32/30 and 14 kDa subunits, collectively known as RPA. RPA is a single stranded DNA binding protein, whose DNA binding activity is believed to reside entirely in the 70 kDa subunit. The complex is required for almost all aspects of cellular DNA metabolism such as DNA replication (1-3), recombination, cell cycle and DNA damage checkpoints, and all major types of DNA repair including nucleotide excision, base excision, mismatch and double-strand break repairs (4-7). In response to genotoxic stress in eukaryotic cells, RPA has been shown to associate with the Rad9/Rad1/Hus1 (9-1-1) checkpoint complex (8). RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (9-11). Phosphorylation of RPA32 occurs at serines 4, 8 and 33 (11). Hyperphosphorylation may alter RPA-DNA and RPA-protein interactions. In addition to the checkpoint partners, RPA interacts with a wide variety of protein partners, including proteins required for normal replication such as RCF, PCNA and Pol α, and also proteins involved in SV40 replication, such as DNA polymerase I and SV40 large T antigen (10,12).

  1. Liu, V.F. and Weaver, D.T. (1993) Mol. Cell Biol. 13, 7222-31.
  2. Wobbe, C.R. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1834-8.
  3. Fairman, M.P. and Stillman, B. (1988) EMBO J. 7, 1211-8.
  4. Wold, M.S. and Kelly, T. (1988) Proc. Natl. Acad. Sci. USA 85, 2523-7.
  5. Zhou, B.B. and Elledge, S.J. (2000) Nature 408, 433-9.
  6. Kastan, M.B. and Bartek, J. (2004) Nature 432, 316-23.
  7. Sancar, A. et al. (2004) Annu. Rev. Biochem. 73, 39-85.
  8. Guo, S. et al. (2006) J Biol Chem 281, 21607-16.
  9. Wu, X. et al. (2005) Oncogene 24, 4728-35.
  10. Binz, S.K. et al. DNA Repair (Amst) 3, 1015-24.
  11. Nuss, J.E. et al. (2005) Biochemistry 44, 8428-37.
  12. Yuzhakov, A. et al. (1999) EMBO J. 18, 6189-99.

Application References

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