Anti-human DNA polymerase δ catalytic subunit/p125 (PolD1) antibody, mousemonoclonal (8A5E3)
Product code70-051

Size50 µg

Storage-20℃

Concentration

1.0 mg/ml

Buffer

PBS- with 50% glycerol

PurityPurified IgG fraction with protein A from hybridoma cell culture medium

ImmunogenPurified recombinant human PolD1 polypeptide from 247 to 1107aa

Isotypemouse IgG2bκ (Some contamination of IgM signal)

Reactivity

human PolD1 protein. Other species have not been tested.

Special notes

Specificity has been validated by western blotting with immunoprecipitatedsamples (Fig. 2 )Application

1. Western blotting (0.33-1 µg/ml ) Fig.1
2. Immunoprecipitation (2-6 µg/µl Ab capture beads). This antibody canprecipitate Polδ complex from human 293T cell lysate (Fig. 2).

BackgroundDNA polymerase δ (Pol δ) is one of the eukaryotic B-family polymerases andinvolved in DNA repair and chromosomal DNA replication. It is composed of alarge catalytic subunit encoded by POLD1 and three accessory subunits;

POLD2, POLD3, and POLD4, which encode proteins p125, p50, p66, and p12,
respectively. The 125kDa catalytic subunit possesses both polymerase and 3' to5' exonuclease activity and is able to synthesize DNA strand with high fidelity
in the presence of its processivity factor PCNA (proliferating cell nuclearantigen). In the replication fork, starting with the low fidelity Pol α synthesizinga ~30 nt RNA/DNA initiator primer, Pol δ synthesizes a major part of 100 ~200
nucleotide (nt) length-lagging strands, Okazaki fragments, discontinuously,
which are then ligated to form the contiguous lagging strand. For synthesis ofeach Okazaki fragment, Replication factor C (RFC) loads PCNA at theprimer/template (P/T) junction. PCNA encircles the duplex DNA and tethers Pol
δ complex to the P/T junction. Mutations in this gene have been associated withvarious cancers and immunodeficiency in human cells.
Data Link

UniProt P28340（DPOD1_HUMAN）

Please note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES. NOT FOR MILITARY USE.

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Data Images: 70-051 Anti-human DNA polymerase δ catalytic subunit/p125 (PolD1) antibody, mousemonoclonal (8A5E3)

Fig 1. Western blotting of 293T cell lysates with 8A5E3
Indicated amounts (μl) of 293T cell lysate (mock; 13μg protein/μl) or 293T cell lysate expressing PolD1,2, 3 and 4 subunits (PolD; 9.8μg protein/μl) were electrophoresed in a 12.5% PAAG and transferred
to a nylon filter with a semidry blotter. Note that this expressing PolD1 protein has hmAzami-Greentag (mAG) and appears about a 150kDa peptide. This filter was masked with 5% skim milk and thep125 peptide was detected with 0.33μg/ml 8A5E3 in CANGET signal Sol.1, 0.2 μg/ml anti-mIgG-H+LHRP (ab205719) in CANGET signal Sol.2 and ImmunoSTAR zeta

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Fig. 2 Immunoprecipitation of Pol δ complex with 8A5E3.
5μl Ab-cap beads were bound with 8A5E3 30 μg. 2 μl each of the beads was mixed with 0.65mg of295T lysate (Mock) or 0.2mg of 293T lysate expressed with mAG-Polδ complex (PolD), respectively.
After washing with PBS containing 10% glycerol, 1mM EDTA and 0.1%NP40, the beads weresuspended with 20μl of SDS sample buffer. (A) 3 or 1μl of the samples (input, unbound, and bound)
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https://www.bioacademia.co.jp/en/were electrophoresed and blotted to nylon filter. Mock input (39μg) and PolD input (9.8μg) were used.
PolDp125 peptide was detected with 0.33 μg/ml 8A5E3 in CANGET signal Sol.1, 0.2 μg/ml anti-mIgGH+L-HRP (ab205719) in CANGET signal Sol.2 and ImmunoSTAR zeta. (B) PolDp66 was detected bywestern blotting with anti-human PolDp66 monoclonal antibody (2A1C11, #70-052) in the same
precipitates as (A). (C) 3μl of the bound samples in the same experiment were electrophoresed andstained with silver. Bands with asterisks are mouse IgG heavy or light chains.
References : This product came from references 3
1. Hindges R and Hubscher U “DNA polymerase delta, an essential enzyme for DNA transactions”

Biol Chem 378: 345-362 (1997) PMID: 9191022
2. Johnson A and O’Donnell M “Cellular DNA replicases: components and dynamics at thereplication fork” Annu Rev Biochem 74: 283-315 (2005) PMID: 15952889
3. Shikata K et al ”The human homologue of fission Yeast cdc27, p66, is a component of active
human DNA polymerase delta” J Biochem 129: 699-708 (200)
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70-052 Anti-human DNA polymerase δ accessory subunit/p66 (PolD3) antibody, mouse monoclonal(2A1C11)

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BioAcademia,Inc.

Tel. 81-6-6877-2335

Fax. 81-6-6877-2336

info@bioacademia.co.jp

https://www.bioacademia.co.jp/en/