To assay foci of Rad52-YFP and Rpa1-YFP, 10 OD of logarithmically growing cells collected at indicated time points after X-ray treatment were fixed in 70% ethanol for 10 min, washed in PBS and then treated with 0.5 mg ml−1 Zymolyase 20 T (ZYMO Research, E1004-A) in PBS for 10 min. Cells were blocked in PBS containing 10% fetal calf serum for 1 h and incubated overnight with a 1:100 dilution of anti-green fluorescent protein antibody (Roche, 1814460001) in blocking buffer containing 0.05% Tween-20. Cells were then washed in PBS and incubated with anti-mouse-FITC at 1:200 in blocking buffer containing 0.05% Tween-20 for 2 h. After washing and resuspension in PBS, cells were applied to coverslips for microscopy. DNA was visualized with 4′,6-diamidino-2-phenylindole. Three independent assays were carried out and 100 cells from more than three fields were counted for each experiment. Foci were observed and counted using an Olympus BX51 microscope and Image-Pro plus 6.0 software.
Zymolyase 20t
Manufactured by Zymo Research
Sourced in United States
Zymolyase 20T is a lyophilized enzyme preparation derived from Arthrobacter luteus. It is designed for the efficient lysis of yeast cell walls, facilitating the extraction and purification of cellular components.
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Lab products found in correlation
4 protocols using zymolyase 20t
1
Quantifying Rad52 and Rpa1 Foci
2
Purification of Yeast Mitochondria
3
Zymolyase Susceptibility Assay
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Susceptibility to Zymolyase was examined as described previously (Charoenbhakdi et al. 2016 (link)). Briefly, log-phase cells were diluted to an OD600 of 0.5 in TE buffer [10 mM Tris–HCl and 1 mM EDTA (pH 7.5)] containing 100 mg L−1 (1 U) Zymolyase 20T (Zymo Research, Orange, CA, USA). The OD600 was measured at 15-min intervals for 2 h by the Wallace Victor 1420 microplate reader (PerkinElmer, Waltham, MA, USA).
4
Isolation and analysis of yeast mitochondria
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Yeast mitochondria were prepared by the method of Herrmann et al. (88 ) using Zymolyase 20T, Zymo Research to obtain spheroplasts. Four milligram of mitochondrial preparation were solubilized in 400 μl of an extraction buffer (20 mM Hepes pH 7.4, 25 mM KCl, 0.5 mM PMSF, 0.8% Triton X100 [or 2% Digitonin], 5 mM MgCl2) and were centrifuged at 27,000g for 15 min and applied onto a linear sucrose gradient 0.3 to 1.0 M, with the same constitution of the correspondent extraction buffer. The linear sucrose gradients were centrifuged for 3 h at 40,000 rpm in the 55Ti Beckmann rotor, Beckmann Coulter (12 (link)). Antibodies and sources are listed in Table S3 .
RNA was extracted from the mitochondria at a protein concentration of 5 mg/ml as described (89 (link)). Total mitochondrial RNAs were separated on a 1% agarose gel. The gel was stained with ethidium bromide, photographed, and the RNAs blotted to a nylon membrane. Following UV crosslinking, the nylon membrane was hybridized overnight at 42 °C with COB and COX1 probes (Table S1 ) that were 3′ end labeled with a biotin kit (Thermo Scientific) as described elsewhere (89 (link)).
RNA was extracted from the mitochondria at a protein concentration of 5 mg/ml as described (89 (link)). Total mitochondrial RNAs were separated on a 1% agarose gel. The gel was stained with ethidium bromide, photographed, and the RNAs blotted to a nylon membrane. Following UV crosslinking, the nylon membrane was hybridized overnight at 42 °C with COB and COX1 probes (
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