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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 |
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KLH-conjugated synthetic peptide derived from known S.cerevisie Sml1 sequence. Gene ID: 854945 |
| antibody format |
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rabbit |
polyclonal |
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serum |
lyophilized |
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| quantity |
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200 µl |
for reconstitution add 200 µl, of sterile water. |
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| storage |
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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 |
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anti-rabbit secondary antibodies |
| additional information |
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to be added when available |
application information
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| recommended dilution |
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1 : 1000 with standard ECL (WB) |
| expected | apparent MW |
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11.83 | 11-12 kDa |
| confirmed reactivity |
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Saccharomyces cerevisiae
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| predicted reactivity |
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Saccharomyces cerevisiae |
| not reactive in |
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no confirmed exceptions from predicted reactivity known at the moment |
| additional information |
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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 |
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Poli et al. (2012). dNTP pools determine fork progression and origin usage under replication stress. The EMBO J. January 2012, 1-12. Tsaponina et al. (2011). Ixr1 Is Required for the Expression of the Ribonucleotide Reductase Rnr1 and Maintenance of dNTP Pools. PLoS Genetics. |
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 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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