Rnr3 | Ribonucleoside-diphosphate reductase large chain 2

AS09 574 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Saccharomyces cerevisiae

Product Information

Immunogen

KLH-conjugated synthetic peptide derived from Saccharomyces cerevisiae Rnr3 protein sequence UniProt: P21672

Host Rabbit

Clonality Polyclonal

Purity Immunogen affinity purified serum in PBS pH 7.4.

Format Lyophilized

Quantity 100 µg

Reconstitution For reconstitution add 100 µl of sterile water

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.

Tested applications Western blot (WB)

Recommended dilution 1 : 500-1 : 1000 (WB)

Expected | apparent MW 97,5 | 98 kDa

Reactivity

Confirmed reactivity Saccharomyces cerevisiae

Predicted reactivity

Saccharomyces cerevisiae

Not reactive in No confirmed exceptions from predicted reactivity are currently known

Application examples

Application example

western blot using anti-Rnr3 antibodies

10 μl total protein from 9.25 x 107 cells of Saccharomyces cerevisiae extracted with 20% TCA as described below were separated on 10% SDS-PAGE and blotted 1.5h (0.5 A) to a nitrocellulose membrane (Whatman PROTRAN BA 85, 0.45 μm). Blots were blocked with 5% non-fat dry milk in TBST for 1.5h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 200 overnight at 4C° with agitation. The antibody solution was decanted and the blot was washed 3 times for 10 min in TBST at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, (AS09 602) diluted to 1:50 000 for 1h at RT with agitation. The blot was washed as above and developed for 3 min with chemiluminescent detection reagent, according to the manufacturers instructions. Exposure time was 10 min.

Courtesy Dr. Andrei Chabes, Umeå University, Sweden

Reactant: Saccharomyces cerevisiae (Yeast)

Application: Western Blotting

Pudmed ID: 21573136

Journal: PLoS Genet

Figure Number: 3A

Published Date: 2011-05-01

First Author: Tsaponina, O., Barsoum, E., et al.

Impact Factor: 5.334

Open Publication

Deletion of IXR1 leads to increased Rnr3 and Rnr4 levels and decreased Sml1 levels.(A) Western blot analysis of Rnr3-HA and Rnr4 levels in wild-type (AC447-2A), ixr1-S366F (TOY619), and ixr1? (TOY621) strains before and after 2 hours treatment with 0.2 mg/L 4-nitroquinoline 1-oxide (4-NQO), 200 mM HU, or 0.02% methyl methanesulfonate (MMS). Rnr3 and Rnr4 levels were quantified in relative units (RU, levels of Rnr3 or Rnr4 divided by the levels of tubulin in corresponding sample) as described in Materials and Methods. ND – not detected. (B) Western blot analysis of Rnr2 levels in wild-type (AC447-2A) and ixr1? (TOY621) strains before treatment and after 2 hours treatment with 0.2 mg/L 4-NQO, 0.02% MMS, or 200 mM HU. (C) Western blot analysis of Sml1 levels in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (D) Western blot analysis of Rad53 phosphorylation status in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (E) Deletion of RAD53 but not of SML1 or DUN1 abolishes the upregulation of Rnr3 and Rnr4 levels in ixr1?. Western blot analysis of Rnr3-HA and Rnr4 levels. The following strains were analyzed: wt (AC447-2A), ixr1? (TOY732), ixr1? sml1? (TOY778), ixr1? dun1? sml1? (TOY772), and ixr1? rad53? sml1? (TOY781).

Additional information

Load per well was approx 3x10^6 cells (of a total extract)

Background

Saccharomyces cerevisiae Rnr3 is catalyzing the biosynthesis of deoxyribonucelaotides. Alternative names: Ribonucleotide reductase large subunit 2, ribonucleotide reductase DNA damage-inducible regulatory subunit 2, ribonucleotide reductase R1 subunit 2

Product citations

Selected references Ajazi et al. (2021) Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis. Dev Cell. 2021 Sep 27;56(18):2607-2622.e6. doi: 10.1016/j.devcel.2021.08.019. Epub 2021 Sep 16. PMID: 34534458.
Cerritelli et al. (2020). High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase. Nucleic Acids Res
Sampaio-Marques et al. (2019). ?-Synuclein toxicity in yeast and human cells is caused by cell cycle re-entry and autophagy degradation of ribonucleotide reductase 1. Aging Cell. 2019 Aug;18(4):e12922. doi: 10.1111/acel.12922.
Schmidt et al. (2019). Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio. Nucleic Acids Res. 2019 Oct 24. pii: gkz1006. doi: 10.1093/nar/gkz1006.
Lafuente-Barquero et al. (2017). The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage. PLOS Genetics, December 27, 2017, doi.org/10.1371/journal.pgen.1007136
Schmidt et al. (2017). Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis. Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4442-E4451. doi: 10.1073/pnas.1618714114.
Graf et al. (2017). Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle. Cell. 2017 Jun 29;170(1):72-85.e14. doi: 10.1016/j.cell.2017.06.006.
Williams et al. (2017). The role of RNase H2 in processing ribonucleotides incorporated during DNA replication. DNA Repair (Amst). 2017 Mar 6. pii: S1568-7864(16)30431-1. doi: 10.1016/j.dnarep.2017.02.016.

Confirmed reactivity:	Saccharomyces cerevisiae
predicted reactivity:	Saccharomyces cerevisiae
not reactive in:	No confirmed exceptions from predicted reactivity are currently known
More images:	Reactant: Saccharomyces cerevisiae (Yeast) Application: Western Blotting Pudmed ID: 21573136 Journal: PLoS Genet Figure Number: 3A Published Date: 2011-05-01 First Author: Tsaponina, O., Barsoum, E., et al. Impact Factor: 5.334 Open Publication Deletion of IXR1 leads to increased Rnr3 and Rnr4 levels and decreased Sml1 levels.(A) Western blot analysis of Rnr3-HA and Rnr4 levels in wild-type (AC447-2A), ixr1-S366F (TOY619), and ixr1? (TOY621) strains before and after 2 hours treatment with 0.2 mg/L 4-nitroquinoline 1-oxide (4-NQO), 200 mM HU, or 0.02% methyl methanesulfonate (MMS). Rnr3 and Rnr4 levels were quantified in relative units (RU, levels of Rnr3 or Rnr4 divided by the levels of tubulin in corresponding sample) as described in Materials and Methods. ND – not detected. (B) Western blot analysis of Rnr2 levels in wild-type (AC447-2A) and ixr1? (TOY621) strains before treatment and after 2 hours treatment with 0.2 mg/L 4-NQO, 0.02% MMS, or 200 mM HU. (C) Western blot analysis of Sml1 levels in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (D) Western blot analysis of Rad53 phosphorylation status in wild-type (W1588-4C) and ixr1? (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (E) Deletion of RAD53 but not of SML1 or DUN1 abolishes the upregulation of Rnr3 and Rnr4 levels in ixr1?. Western blot analysis of Rnr3-HA and Rnr4 levels. The following strains were analyzed: wt (AC447-2A), ixr1? (TOY732), ixr1? sml1? (TOY778), ixr1? dun1? sml1? (TOY772), and ixr1? rad53? sml1? (TOY781).
Picture (footer):	Application example 10 μl total protein from 9.25 x 107 cells of Saccharomyces cerevisiae extracted with 20% TCA as described below were separated on 10% SDS-PAGE and blotted 1.5h (0.5 A) to a nitrocellulose membrane (Whatman PROTRAN BA 85, 0.45 μm). Blots were blocked with 5% non-fat dry milk in TBST for 1.5h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 200 overnight at 4C° with agitation. The antibody solution was decanted and the blot was washed 3 times for 10 min in TBST at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, (AS09 602) diluted to 1:50 000 for 1h at RT with agitation. The blot was washed as above and developed for 3 min with chemiluminescent detection reagent, according to the manufacturers instructions. Exposure time was 10 min. Courtesy Dr. Andrei Chabes, Umeå University, Sweden
calculated \| apparent molecular mass [kDa]:	97,5 \| 98 kDa
Clonality:	Polyclonal
Format:	Lyophilized
Host:	Rabbit
immunogen:	KLH-conjugated synthetic peptide derived from Saccharomyces cerevisiae Rnr3 protein sequence UniProt: P21672
Purity:	Immunogen affinity purified serum in PBS pH 7.4.
Quantity:	100 µg
recommended dilution:	1 : 500-1 : 1000 (WB)
Reconstitution:	For reconstitution add 100 µl of sterile water
storage:	Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
tested applications:	Western blot (WB)
additional information (application):	Load per well was approx 3x10^6 cells (of a total extract)
All references:	Ajazi et al. (2021) Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis. Dev Cell. 2021 Sep 27;56(18):2607-2622.e6. doi: 10.1016/j.devcel.2021.08.019. Epub 2021 Sep 16. PMID: 34534458. Cerritelli et al. (2020). High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase. Nucleic Acids Res Sampaio-Marques et al. (2019). ?-Synuclein toxicity in yeast and human cells is caused by cell cycle re-entry and autophagy degradation of ribonucleotide reductase 1. Aging Cell. 2019 Aug;18(4):e12922. doi: 10.1111/acel.12922. Schmidt et al. (2019). Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio. Nucleic Acids Res. 2019 Oct 24. pii: gkz1006. doi: 10.1093/nar/gkz1006. Lafuente-Barquero et al. (2017). The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage. PLOS Genetics, December 27, 2017, doi.org/10.1371/journal.pgen.1007136 Schmidt et al. (2017). Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis. Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4442-E4451. doi: 10.1073/pnas.1618714114. Graf et al. (2017). Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle. Cell. 2017 Jun 29;170(1):72-85.e14. doi: 10.1016/j.cell.2017.06.006. Williams et al. (2017). The role of RNase H2 in processing ribonucleotides incorporated during DNA replication. DNA Repair (Amst). 2017 Mar 6. pii: S1568-7864(16)30431-1. doi: 10.1016/j.dnarep.2017.02.016.
background:	Saccharomyces cerevisiae Rnr3 is catalyzing the biosynthesis of deoxyribonucelaotides. Alternative names: Ribonucleotide reductase large subunit 2, ribonucleotide reductase DNA damage-inducible regulatory subunit 2, ribonucleotide reductase R1 subunit 2

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