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Sml1 | Suppressor of Mec1 lethality

AS10 847 | Clonality: Polyclonal  |  Host: Rabbit  |  Reactivity:Saccharomyces cerevisiae

Sml1 | Suppressor of Mec1 lethality in the group Antibodies, Bacterial/Fungal at Agrisera AB (Antibodies for research) (AS10 847)

DATA SHEET IN PDF

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How to cite this product:
Product name, number (Agrisera, Sweden)

Data sheet Product citations Protocols Add review

Product Information

Immunogen

KLH-conjugated synthetic peptide derived from known S.cerevisie Sml1 sequence.  Gene ID: 854945

Host Rabbit
Clonality Polyclonal
Purity Serum
Format Lyophilized
Quantity 50 µl
Reconstitution For reconstitution add 50 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 tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Western blot (WB)
Recommended dilution 1 : 1000 (WB)
Expected | apparent MW

11.83 | 11-12 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 examples

Application example

western blot detection of Sml1

 

10 μl total protein from 9.25 x 107 cells of Saccharomyces cerevisiae extracted with 20% TCA as described below were separated on 20% 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: 5 000 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 from Agrisera, (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. Exposure time was 30 seconds.
 
Courtesy Dr. Andrei Chabes, Umeå University, Sweden

Additional information

Cell preparation for western blot: cells were harvested by centrifugation (4000 x g , 6 min, 4 °C). Supernatant was discarded and cells were resuspended in 500 μl cold TCA buffer (20 mM Tris, pH 8, 50 mM ammonium acetate, 2 mM EDTA, 1 tablet/10 ml of Complete Mini Protease inhibitor cocktail with EDTA (Roche Diagnostics GmbH)). 500 μl 0.5 mm Zirconia/Silica Beads (BioSpec Products, Inc. 11079105z) and 500 μl cold 20 % TCA was added. Samples were vigorously vortexed twice for 30 sec (kept on ice in between). 750 μl from the liquid phase was transferred into a fresh Eppendorf tube. Samples were centrifuged for 10 min (20000 x g, 4 °C). The pellet was resuspended in 300 μl TCA-Laemmli buffer and boiled for 10 min at 100 °C.

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Background

Background

Sml1 is a ribonucleotide reductase inhibitor involved in regulation of dNTP production. It is regulated by Mec1p and Rad53p during DNA damage and S phase. Synonymes: Yml058w.

Product citations

Selected references 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
Corcoles-Saez et al. (2019). Essential Function of Mec1, the Budding Yeast ATM/ATR Checkpoint-Response Kinase, in Protein Homeostasis. Dev Cell. 2018 Aug 20;46(4):495-503.e2. doi: 10.1016/j.devcel.2018.07.011.
Garbacz et al. (2019). The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity. Nucleic Acids Res. 2019 Jan 30. doi: 10.1093/nar/gkz048.
Golla et al. (2017). A systematic assessment of chemical, genetic, and epigenetic factors influencing the activity of anticancer drug KP1019 (FFC14A). Oncotarget. 2017 Sep 30;8(58):98426-98454. doi: 10.18632/oncotarget.21416.
Dmowski et al. (2017). Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint. PLoS Genet. 2017 Jan 20;13(1):e1006572. doi: 10.1371/journal.pgen.1006572.
Mertz et al. (2015). Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity. Proc Natl Acad Sci U S A. 2015 May 12;112(19):E2467-76. doi: 10.1073/pnas.1422934112. Epub 2015 Mar 31.
Singh et al. (2014). Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae. FEBS Lett. 2014 Feb 20. pii: S0014-5793(14)00137-9. doi: 10.1016/j.febslet.2014.02.017.
Azad et al. (2013). Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in Saccharomyces cerevisiae. PLoS One, March 8.
Poli et al. (2012).dNTP pools determine fork progression and origin usage under replication stress. The EMBO J. January 2012, 1-12.

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