Sml1 | Suppressor of Mec1 lethality

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


product information
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.
Immunogen		KLH-conjugated synthetic peptide derived from known S.cerevisie Sml1 sequence. Gene ID: 854945
Host		Rabbit
Clonality		Polyclonal
Clone
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)
Related products		anti-rabbit secondary antibodies
Additional information

application information
Recommended dilution		1 : 1000 (WB)
Expected \| apparent MW		11.83 \| 11-12 kDa
Confirmed reactivity		Saccharomyces cerevisiae
Predicted reactivity		Saccharomyces cerevisiae
Not reactive in		No confirmed exceptions from predicted reactivity are currently known.
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.
Selected references		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.

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 SuperSignal West Pico Stable Solution (1856135 Thermo Scientific) and SuperSignal West Pico Luminol/Enhancer Solution (1856136 Thermo Scientific) mixed 1:1 rate according to the manufacturers instructions. Exposure time was 30 seconds.

Courtesy Dr. Andrei Chabes, Umeå University, Sweden

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