Anti-human DNA polymerase δ accessory subunit/p66 (PolD3) antibody, mousemonoclonal (2A1C11)
Product code70-052

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 full-length PolD3 protein

Isotypemouse IgG2bκ

Reactivity

human PolD3 protein. Other species have not been tested.

Special notes

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

1. Western blotting (0.33-1µg/ml ) Fig.1
2. Immunoprecipitation (2-6µg/µl Ab capture beads). This antibody can
precipitate 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. p66 plays a role inregulating the activity of Pol δ through interactions with other subunits andPCNA.
Data Link

UniProtKB/Swiss-Prot Q15054 (DPOD3_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: 70-052 Anti-human DNA polymerase δ accessory subunit/p66 (PolD3) antibody, mouse

PolD 0.3
PolD 1

Mock 0.3
Mock 1
Mock 3

monoclonal (2A1C11)

150→
100→
75→
50→
37→

Fig 1. Western blotting of 293T cell lysates with 2A1C11
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. This filter was masked with 5% skim milk and the p66 peptide
was detected with 0.33μg/ml 2A1C11 in CANGET signal Sol.1, 0.2μg/ml anti-mIgG-H+L-HRP(ab205719) in CANGET signal Sol.2 and ImmunoSTAR zeta. Inserted box is the longer exposure of150→
100→
75→
50→150→←66**100→75→←125+mAG←66*37→50→(B)

37→

150→100→75→25→50→

2A1C11 PolD

2A1C11 beads(C)

Mock input
Mock UnboundMock Bound
PolD input
PolD Unbound
PolD Bound(A)

2A1C11 Mockthe 50-100kDa area.

←50*

20→*

*

15→

←1237→

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Fig. 2 Immunoprecipitation of Pol δ complex with 2A1C11.
10µl of Ab-cap beads were prebound with 30μg of 2A1C11. 4µl each of the beads was mixed with0.65mg of 295T mock lysate (Mock) or 0.2mg of 293T lysate expressed with mAG-PolD complex (PolD),respectively.
Note that this expressing PolD1 protein has hmAzami-Green tag (mAG) and appears about a 150kDapeptide. After washing with PBS containing 10% glycerol, 1mM EDTA and 0.1%NP40, the beads weresuspended with 20µl of SDS sample buffer.
3µl (Mock) or 1µl (PolD) of the samples (input, unbound, and bound) were electrophoresed in 12.5%
PAAG and blotted to nylon filter. Mock input (39µg) and PolD input (9.8µg) were used. (A) PolDp66
peptide was detected with 0.33µg/ml 2A1C11 in CANGET signal Sol.1. (B) PolDp125 peptide or
150kDa peptide of p125 with mAG-tag were detected with 0.33µg/ml 8A5E3 (E70-051) in CANGET
signal Sol.1. Both antibodies were detected with 0.2µg/ml anti-mIgG-H+L-HRP (ab205719) inCANGET signal Sol.2 and ImmunoSTAR Z. (C) 3μl of the bound samples (Mock and PolD) of theimmunoprecipitation were electrophoresed with the beads-bound antibody (Blank), and stained withsilver. 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)
Related products:
70-051 Anti-human DNA polymerase δ catalytic subunit/p125 (PolD1) antibody, mouse monoclonal(8A5E3)70-053 Anti-human DNA polymerase δ accessory subunit/p66(PolD3) recombinant antibody withmouse Fc (r2A1C11LvR31)

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BioAcademia,Inc.

Tel. 81-6-6877-2335

Fax. 81-6-6877-2336

info@bioacademia.co.jp

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