Recombinant
RabMAb

Recombinant Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394)

Rabbit recombinant monoclonal RPA32/RPA2 (phospho T21) antibody [EPR2846(2)]. Validated in WB, IP, Dot and tested in Mouse, Rat, Human. Cited in 16 publication(s). Independently reviewed in 1 review(s).

Overview

  • Product name

    Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)]
    See all RPA32/RPA2 primary antibodies
  • Description

    Rabbit monoclonal [EPR2846(2)] to RPA32/RPA2 (phospho T21)
  • Host species

    Rabbit
  • Specificity

    ab109394 only detects RPA32/RPA2 phosphorylated at threonine 21. The antibody has been tested with milk (5%) and BSA (2%) as blocking reagents, but it provided better results with 5%milk.

    This antibody cross-reactivates with non-phospho substrate in specific situations.

  • Tested applications

    Suitable for: Dot blot, WB, IPmore details
    Unsuitable for: Flow Cyt,ICC/IF or IHC-P
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human RPA32/RPA2 (phospho T21). The exact sequence is proprietary.

  • Positive control

    • WB: HeLa whole cell lysate treated with Calyculin A or Camptothecin. Dot Blot: RPA32/RPA2 (pT21) phospho peptide.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab109394 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Dot blot 1/1000.
WB 1/50000 - 1/200000. Detects a band of approximately 32 kDa (predicted molecular weight: 29 kDa).

The antibody has been tested with milk (5%) and BSA (2%) as blocking reagents, but it provided better results with 5%milk.

IP 1/10 - 1/100.
  • Application notes
    Is unsuitable for Flow Cyt,ICC/IF or IHC-P.
  • Target

    • Function

      Required for DNA recombination, repair and replication. The activity of RP-A is mediated by single-stranded DNA binding and protein interactions.
      Functions as component of the alternative replication protein A complex (aRPA). aRPA binds single-stranded DNA and probably plays a role in DNA repair; it does not support chromosomal DNA replication and cell cycle progression through S-phase. In vitro, aRPA cannot promote efficient priming by DNA polymerase alpha but supports DNA polymerase delta synthesis in the presence of PCNA and replication factor C (RFC), the dual incision/excision reaction of nucleotide excision repair and RAD51-dependent strand exchange.
    • Post-translational
      modifications

      Phosphorylated in a cell-cycle-dependent manner (from the S phase until mitosis). Phosphorylated by ATR upon DNA damage, which promotes its translocation to nuclear foci. Can be phosphorylated in vitro by PRKDC/DNA-PK in the presence of Ku and DNA, and by CDK1.
    • Cellular localization

      Nucleus. Nucleus > PML body. Also present in PML nuclear bodies. Redistributes to discrete nuclear foci upon DNA damage.
    • Information by UniProt
    • Database links

    • Alternative names

      • 60S acidic ribosomal protein P1 antibody
      • AA409079 antibody
      • AI325195 antibody
      • AU020965 antibody
      • ik:tdsubc_2g1 antibody
      • M(2)21C antibody
      • MGC137236 antibody
      • OTTHUMP00000004008 antibody
      • p32 antibody
      • p34 antibody
      • RCJMB04_6d17 replication protein A2, 32kDa antibody
      • REPA2 antibody
      • Replication factor A protein 2 antibody
      • Replication protein A 32 kDa subunit antibody
      • Replication protein A 32kDa subunit antibody
      • Replication protein A 34 kDa subunit antibody
      • Replication protein A antibody
      • Replication Protein A2 (32kDa) antibody
      • Replication protein A2 antibody
      • Replication protein A2, 32kDa antibody
      • RF-A protein 2 antibody
      • Rf-A2 antibody
      • RFA antibody
      • RFA2_HUMAN antibody
      • RP-A p32 antibody
      • RP-A p34 antibody
      • RP21C antibody
      • RPA 2 antibody
      • RPA 32 antibody
      • RPA antibody
      • Rpa2 antibody
      • RPA32 antibody
      • RPA34 antibody
      • RpLP1 antibody
      • RpP2 antibody
      • xx:tdsubc_2g1 antibody
      • zgc:109822 antibody
      see all

    Images

    • All lanes : Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394) at 1/5000 dilution

      Lane 1 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates
      Lane 2 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates. Then the membrane was incubated with phosphatase.

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 29 kDa
      Observed band size: 32,36 kDa
      why is the actual band size different from the predicted?


      Exposure time: 30 seconds


      Blocking/Diluting buffer and concentration: 5% NFDM/TBST

    • Direct ELISA antigen dose-response curve using purified ab109394.

      Primary antibody: ab109394 (0-1000nweg/ml).

      Antigens: P1: RPA32/RPA2 (phospho T21) at 10ng/ml and 1ng/ml; NP: non-phospho at 10ng/ml and 1 ng/ml.

      Secondary antibody: Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) at 1/2500 dilution.

    • Dot blot analysis of c-Myb RPA32/RPA2 (pT21) phospho peptide (Lane 1) and RPA32/RPA2 non-phospho peptide (Lane 2) labeling RPA32/RPA2 (phospho T21) with purified ab109394 at a dilution of 1/5000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/2500.

      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • All lanes : Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394) at 1/2000 dilution

      Lane 1 : C6 (Rat glial tumor glial cell) whole cell lysates
      Lane 2 : C6 (Rat glial tumor glial cell) treated with 100nM calyculin A for 60 minutes whole cell lysates
      Lane 3 : C6 (Rat glial tumor glial cell) treated with 100nM calyculin A for 60 minutes whole cell lysates.Then the membrane was incubated with phosphatase.

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 29 kDa
      Observed band size: 32,36 kDa why is the actual band size different from the predicted?



      Blocking/Diluting buffer and concentration:5% NFDM/TBST.

      Exposure time: 110 seconds.

    • All lanes : Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394) at 1/2000 dilution

      Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
      Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) treated with 100ng/ml calyculin A for 30 minutes whole cell lysates
      Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) treated with 100ng/ml calyculin A for 60 3minutes whole cell lysates.Then the membrane was incubated with phosphatase

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 29 kDa
      Observed band size: 32,36 kDa why is the actual band size different from the predicted?



      Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

      Exposure time: 180 seconds.

    • All lanes : Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394) at 1/50000 dilution

      Lane 1 : HeLa whole cell lysate - untreated
      Lane 2 : HeLa whole cell lysate - treated with Calyculin A
      Lane 3 : HeLa whole cell lysate - treated with Calyculin A and Alkaline Phosphatase

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 29 kDa
      Observed band size: 32 kDa why is the actual band size different from the predicted?


      Exposure time: 30 seconds


      Blocking and dilution buffer: 5% NFDM/TBST.

    • Dot blot analysis of RPA32/RPA2 (pT21) phospho peptide (lane 1) and RPA32/RPA2 non-phospho peptide (lane 2) labelling RPA32/RPA2 (phospho T21) with ab109394 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • All lanes : Anti-RPA32/RPA2 (phospho T21) antibody [EPR2846(2)] (ab109394) at 1/50000 dilution

      Lane 1 : HeLa cell lysates, untreated
      Lane 2 : HeLa cell lysates, treated with Camptothecin

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 29 kDa
      Observed band size: 32 kDa why is the actual band size different from the predicted?

    References

    This product has been referenced in:

    • Witkiewicz AK  et al. Targeting the Vulnerability of RB Tumor Suppressor Loss in Triple-Negative Breast Cancer. Cell Rep 22:1185-1199 (2018). WB . Read more (PubMed: 29386107) »
    • Menolfi D  et al. Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA. Nat Commun 9:5351 (2018). Read more (PubMed: 30559436) »
    See all 17 Publications for this product

    Customer reviews and Q&As

    This product is known to not work in this application or species.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (U2OS)
    Permeabilization
    Yes - Triton 0.3%
    Specification
    U2OS
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Jan 24 2013

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