Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

DNA Replication Antibody Sampler Kit #8341

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
CDT1 Antibody #3386 40 µl W H 65 Rabbit
MCM2 (D7G11) XP® Rabbit mAb #3619 40 µl W IP IHC-P IF-IC ChIP H M R Mk 125 Rabbit IgG
MCM3 (D47B6) Rabbit mAb #4003 40 µl W H M R Mk 100 Rabbit IgG
MCM7 (D10A11) XP® Rabbit mAb #3735 40 µl W IP IHC-P IF-IC H M R Hm Mk Dg 80 Rabbit IgG
PCNA (PC10) Mouse mAb #2586 40 µl W IP IHC-P IF-IC F H M R Mk B Pg 36 Mouse IgG2a
RPA70 (C24F2) Rabbit mAb #2193 40 µl W H Mk 70 Rabbit IgG
p58 Primase (8D3) Rat mAb #4726 40 µl W IP H M R Hm Mk 58 Rat IgG2a
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse
Anti-rat IgG, HRP-linked Antibody #7077 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

All antibodies recognize endogenous levels of the respective target protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using RPA70 (C24F2) Rabbit mAb #2193.

Western Blotting

Western Blotting

Western blot analysis of human (HeLa), murine (SV-T2), bovine (BAEC), and porcine (PAE) cell extracts using PCNA (PC10) Mouse mAb #2586.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using CDT1 Antibody #3386.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using MCM2 (D7G11) XP® Rabbit mAb #3619.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using MCM7 (D10A11) XP® Rabbit mAb #3735.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using MCM3 (D47B6) Rabbit mAb #4003.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using p58 Primase (8D3) Rat mAb #4726.

Description

The DNA Replication Antibody Sampler Kit provides a fast and economical means of evaluating multiple targets regulating DNA replication. The kit contains enough primary antibodies to perform four western blots with each antibody.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near: the amino terminus of human MCM2 protein, the amino terminus of human MCM3 protein, the carboxy terminus of human MCM7 protein, or residues surrounding Thr164 of human RPA70 protein. Monoclonal antibodies are produced by immunizing animals with: a recombinant Protein A-PCNA fusion protein or full-length human recombinant p58 primase. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human CDT1 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The initiation of DNA replication in mammalian cells is a highly coordinated process that is regulated by several protein complexes. Origins of replication (ORCs), at which replication is initiated, are dispersed throughout the genome. Their activities are regulated via the sequential binding of pre-replication and replication factors that initiate formation of replication forks, the active structures at which DNA is synthesized. The origin recognition complex is thought to be bound to chromatin throughout the cell cycle (1,2). The pre-replication complex (Pre-RC) forms in late mitosis/early G1 phase beginning with the binding of CDT1 and CDC6 to the origin. Together CDT1 and CDC6 promote the loading of the heterohexameric minichromosome maintenance (MCM) complex. This process is referred to as chromatin licensing. Licensing of the chromatin permits the DNA to replicate only once per cell cycle, helping to ensure that genetic alterations and malignant cell growth do not occur (reviewed in 3). The canonical MCM complex proteins (MCM2-7) are a family of six related phospho-proteins that function, in part, as the eukaryotic replicative DNA helicase (3,4). Phosphorylation and ubiquitination of the MCM2, MCM3, MCM4, and MCM6 subunits appears to regulate MCM complex activity and the initiation of DNA synthesis (5-7). MCM proteins are removed during DNA replication, causing chromatin to become unlicensed, inhibiting Pre-RC reformation. In addition to DNA polymerase, initiation of DNA replication requires a pair of primase subunits. DNA Primase activity catalyzes de novo synthesis of an RNA/DNA primer (initiator DNA) on the leading and lagging strands, while polymerase activity extends the initiator DNA (8). The 48 and 58 kDa primase subunits cooperate in the synthesis of small RNA primers. p48 is the catalytically active subunit (9), while p58 couples p48 to the polymerase to allow the transfer of primers to the active site. The p58 subunit may also play a role in regulation of primer length (10,11). Once replication is initiated, Proliferating Cell Nuclear Antigen (PCNA) serves as an accessory factor for DNA polymerases delta and epsilon, acting to tether these polymerases to template DNA during replication. Interactions of PCNA with DNA polymerases increase the processivity of leading strand synthesis. PCNA, a member of DNA sliding clamp family, is a homotrimeric ring complex that encircles and slides along the DNA double helix as the replication fork progresses (12). Multiple proteins involved in DNA replication, DNA repair, and cell cycle control bind to PCNA and regulate DNA synthesis. PCNA is loaded onto the DNA in an ATP-dependent manner by a multiprotein clamp loader, Replication Factor C (RFC) (13). RFC, in turn, associates with DNA via interactions with the single-stranded DNA binding protein complex, Replication Protein A (RPA). The canonical RPA complex is heterotrimeric and composed of RPA1 (RPA70), RPA2 (RPA32), and RPA3 (RPA14) subunits. RPA recognizes and stabilizes single stranded DNA in repair processes and DNA recombination, and plays a role in replication (14-17).

  1. Okuno, Y. et al. (2001) EMBO J 20, 4263-77.
  2. McNairn, A.J. et al. (2005) Exp Cell Res 308, 345-56.
  3. Forsburg, S.L. (2004) Microbiol Mol Biol Rev 68, 109-31.
  4. Johnson, A. and O'Donnell, M. (2005) Annu Rev Biochem 74, 283-315.
  5. Charych, D.H. et al. (2008) J Cell Biochem 104, 1075-86.
  6. Masai, H. et al. (2006) J Biol Chem 281, 39249-61.
  7. Lin, D.I. et al. (2008) Proc Natl Acad Sci U S A 105, 8079-84.
  8. Shiratori, A. et al. (1995) Genomics 28, 350-3.
  9. Copeland, W.C. (1997) Protein Expr Purif 9, 1-9.
  10. Copeland, W.C. and Wang, T.S. (1993) J Biol Chem 268, 26179-89.
  11. Arezi, B. and Kuchta, R.D. (2000) Trends Biochem Sci 25, 572-6.
  12. Bowman, G.D. et al. (2004) Nature 429, 724-30.
  13. Zhang, G. et al. (1999) Proc Natl Acad Sci U S A 96, 1869-74.
  14. Sakaguchi, K. et al. (2009) FEBS J 276, 943-63.
  15. Zou, Y. et al. (2006) J Cell Physiol 208, 267-73.
  16. Wold, M.S. (1997) Annu Rev Biochem 66, 61-92.
  17. Binz, S.K. et al. DNA Repair (Amst) 3, 1015-24.

Application References

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Protocols

* Product-specific protocol.

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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