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PCNA Mouse Monoclonal Antibody [A6-G11]

Usd: 350

Specification

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Catalog# EM111201

PCNA Mouse Monoclonal Antibody [A6-G11]

Application

  • WB

  • IHC-P

  • FC

  • IF-Cell

Reactivity

  • Human

  • Mouse

  • Rat

Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

PCNA Mouse Monoclonal Antibody [A6-G11]

Antibody Type

Mouse Monoclonal Antibody

Immunogen

Recombinant protein within human PCNA aa 1-261.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IHC-P, FC, IF-Cell

Molecular Weight

Predicted band size: 29 kDa

Positive Control

HCT 116 cell lysate, HEK-293 cell lysate, Raji cell lysate, HeLa cell lysate, K-562 cell lysate, NIH/3T3 cell lysate, RAW264.7 cell lysate, L-929 cell lysate, C2C12 cell lysate, rat spleen tissue lysate, mouse spleen tissue lysate, human liver tissue lysate, HeLa, human colon cancer tissue, mouse testis tissue, rat testis tissue.

Conjugation

unconjugated

Clone Number

A6-G11

RRID

AB_3068738

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG1

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000-1:5,000

  • IHC-P

  • 1:10,000

  • FC

  • 1:1,000

  • IF-Cell

  • 1:500

Target

Function

Auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'-5' exonuclease and 3'-phosphodiesterase, but not apurinic-apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2. Plays a key role in DNA damage response (DDR) by being conveniently positioned at the replication fork to coordinate DNA replication with DNA repair and DNA damage tolerance pathways. Acts as a loading platform to recruit DDR proteins that allow completion of DNA replication after DNA damage and promote postreplication repair: Monoubiquitinated PCNA leads to recruitment of translesion (TLS) polymerases, while 'Lys-63'-linked polyubiquitination of PCNA is involved in error-free pathway and employs recombination mechanisms to synthesize across the lesion.

Background References

1. "Targeting tyrosine phosphorylation of PCNA inhibits prostate cancer growth.” Zhao,H., et al. Mol. Cancer Ther. 10: 29-36(2011)

2. “A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker.” Linda H. Malkas, Brittney Shea Herbert, Waleed Abdel-Aziz, Lacey E. Dobrolecki, et al. Proc Natl Acad Sci. 103(51)(2006)

Sequence Similarity

Belongs to the PCNA family.

Post-translational Modification

Phosphorylated. Phosphorylation at Tyr-211 by EGFR stabilizes chromatin-associated PCNA.; Acetylated by CREBBP and p300/EP300; preferentially acetylated by CREBBP on Lys-80, Lys-13 and Lys-14 and on Lys-77 by p300/EP300 upon loading on chromatin in response to UV irradiation. Lysine acetylation disrupts association with chromatin, hence promoting PCNA ubiquitination and proteasomal degradation in response to UV damage in a CREBBP- and EP300-dependent manner. Acetylation disrupts interaction with NUDT15 and promotes degradation.; Ubiquitinated. Following DNA damage, can be either monoubiquitinated to stimulate direct bypass of DNA lesions by specialized DNA polymerases or polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Following induction of replication stress, monoubiquitinated by the UBE2B-RAD18 complex on Lys-164, leading to recruit translesion (TLS) polymerases, which are able to synthesize across DNA lesions in a potentially error-prone manner. An error-free pathway also exists and requires non-canonical polyubiquitination on Lys-164 through 'Lys-63' linkage of ubiquitin moieties by the E2 complex UBE2N-UBE2V2 and the E3 ligases, HLTF, RNF8 and SHPRH. This error-free pathway, also known as template switching, employs recombination mechanisms to synthesize across the lesion, using as a template the undamaged, newly synthesized strand of the sister chromatid. Monoubiquitination at Lys-164 also takes place in undamaged proliferating cells, and is mediated by the DCX(DTL) complex, leading to enhance PCNA-dependent translesion DNA synthesis. Sumoylated during S phase.; Methylated on glutamate residues by ARMT1/C6orf211.

Subcellular Location

Nucleus

UNIPROT

SwissProt: P12004 Human

SwissProt: P17918 Mouse

SwissProt: P04961 Rat

Synonyms

ATLD2 antibody

cb16 antibody

Cyclin antibody

DNA polymerase delta auxiliary protein antibody

etID36690.10 antibody

fa28e03 antibody

fb36g03 antibody

HGCN8729 antibody

MGC8367 antibody

Mutagen-sensitive 209 protein antibody

Expand

Images

  • Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/1,000 dilution.<br /><br />Lane 1: HCT 116 cell lysate (20 µg/Lane)<br />Lane 2: HEK-293 cell lysate (20 µg/Lane)<br />Lane 3: Raji cell lysate (20 µg/Lane)<br />Lane 4: HeLa cell lysate (20 µg/Lane)<br />Lane 5: K-562 cell lysate (20 µg/Lane)<br />Lane 6: NIH/3T3 cell lysate (20 µg/Lane)<br /><br />Predicted band size: 29 kDa<br />Observed band size: 34 kDa<br /><br />Exposure time: 1 minutes 2 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) at 1/100,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (EM111201) at 1/1,000 dilution.

    Lane 1: HCT 116 cell lysate (20 µg/Lane)
    Lane 2: HEK-293 cell lysate (20 µg/Lane)
    Lane 3: Raji cell lysate (20 µg/Lane)
    Lane 4: HeLa cell lysate (20 µg/Lane)
    Lane 5: K-562 cell lysate (20 µg/Lane)
    Lane 6: NIH/3T3 cell lysate (20 µg/Lane)

    Predicted band size: 29 kDa
    Observed band size: 34 kDa

    Exposure time: 1 minutes 2 seconds;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM111201) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/100,000 dilution was used for 1 hour at room temperature.

  • Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/1,000 dilution.<br /><br />Lane 1: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 2: RAW264.7 cell lysate (20 µg/Lane)<br />Lane 3: L-929 cell lysate (20 µg/Lane)<br />Lane 4: C2C12 cell lysate (20 µg/Lane)<br />Lane 5: Rat spleen tissue lysate (40 µg/Lane)<br />Lane 6: Mouse spleen tissue lysate (40 µg/Lane)<br />Lane 7: Human liver tissue lysate (40 µg/Lane)<br /><br />Predicted band size: 29 kDa<br />Observed band size: 34 kDa<br /><br />Exposure time: 7 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (EM111201) at 1/1,000 dilution.

    Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 2: RAW264.7 cell lysate (20 µg/Lane)
    Lane 3: L-929 cell lysate (20 µg/Lane)
    Lane 4: C2C12 cell lysate (20 µg/Lane)
    Lane 5: Rat spleen tissue lysate (40 µg/Lane)
    Lane 6: Mouse spleen tissue lysate (40 µg/Lane)
    Lane 7: Human liver tissue lysate (40 µg/Lane)

    Predicted band size: 29 kDa
    Observed band size: 34 kDa

    Exposure time: 7 seconds;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM111201) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/5,000 dilution.<br /><br />Lane 1: HeLa-si NT cell lysate<br />Lane 2: HeLa-si PCNA cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 29 kDa<br />Observed band size: 34 kDa<br /><br />Exposure time: 24 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of PCNA on different lysates with Mouse anti-PCNA antibody (EM111201) at 1/5,000 dilution.

    Lane 1: HeLa-si NT cell lysate
    Lane 2: HeLa-si PCNA cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 29 kDa
    Observed band size: 34 kDa

    Exposure time: 24 seconds;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM111201) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of HeLa cells labeling PCNA with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/500 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 488, <a href="/products/HA1125" style="font-weight: bold;text-decoration: underline;">HA1125</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. beta Tubulin (<a href="/products/ET1602-4" style="font-weight: bold;text-decoration: underline;">ET1602-4</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 594, <a href="/products/HA1122" style="font-weight: bold;text-decoration: underline;">HA1122</a>) were used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of HeLa cells labeling PCNA with Mouse anti-PCNA antibody (EM111201) at 1/500 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-PCNA antibody (EM111201) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

  • Flow cytometric analysis of HeLa cells labeling PCNA.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>, 1μg/mL) (red) compared with Mouse IgG1 Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Mouse IgG Secondary antibody (<a href="/products/HA1125" style="font-weight: bold;text-decoration: underline;">HA1125</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of HeLa cells labeling PCNA.

    Cells were fixed and permeabilized. Then stained with the primary antibody (EM111201, 1μg/mL) (red) compared with Mouse IgG1 Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Mouse IgG Secondary antibody (HA1125) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

  • Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/2,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Mouse anti-PCNA antibody (EM111201) at 1/2,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM111201) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/10,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Mouse anti-PCNA antibody (EM111201) at 1/10,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM111201) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat testis tissue with Mouse anti-PCNA antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/10,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM111201" style="font-weight: bold;text-decoration: underline;">EM111201</a>) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat testis tissue with Mouse anti-PCNA antibody (EM111201) at 1/10,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM111201) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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