Zymolyase

Manufactured by Zymo Research

Sourced in United States, Germany

Zymolyase is an enzymatic preparation derived from the yeast Arthrobacter luteus. It is used for the lysis of yeast cell walls, enabling the release of cellular contents such as nucleic acids and proteins.

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Lab products found in correlation

Mutanolysin, by Merck Group (3 mentions) Lysostaphin, by Merck Group (3 mentions) Lysozyme, by Merck Group (2 mentions) Sorbitol, by Merck Group (2 mentions) Triton x 100, by Serva Electrophoresis (2 mentions) Rabbit monoclonal atp7b antibody, by Abcam (2 mentions) Vectashield mounting medium, by Vector Laboratories (2 mentions) Lysozyme, by MP Biomedicals (2 mentions) Lithium dodecyl sulfate (lds), by Merck Group (2 mentions) Ethylene diamine tetraacetic acid (edta), by Teknova (2 mentions) Ethylene diamine tetraacetic acid (edta), by New England Biolabs (2 mentions) Proteinase k, by New England Biolabs (2 mentions) Sodium chloride (nacl), by Merck Group (2 mentions) Turbo dna free kit, by Thermo Fisher Scientific (2 mentions) Rneasy kit, by Qiagen (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Chitinase, by Merck Group (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Glass fibre gf c filters, by Cytiva (1 mentions)

Spelling variants of this product

Zymolyase 20T (4 citations) Zymolyase-100T (4 citations) R-Zymolyase (3 citations)

43 protocols using zymolyase

Single-cell Transcriptomics of Yeast Cells via Droplet-based Sequencing

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Cells were subjected to Drop-Seq following the original protocol² with the following modifications. Barcoded beads were resuspended in the yeastDrop-Seq solution containing the reagents as shown in Table 1. Cells were diluted to 50 cells/μL. After initial droplet formation, droplets were incubated for 30 min at 30 °C to ensure Zymolyase breakdown of cell walls followed by cell bursting (Supplementary Fig. 1). After incubation, droplet quality was evaluated and >95% droplet-uniformity remained intact.Table 1

yeastDrop-Seq solution.

Solution (total volume)	Reagent	Reagent volume
yeastDrop-seq solution (2223.5 μL)	Zymolyase (Zymo Research E1004), 2 Units/μL	37.5 μL
	Lysis buffer (no DTT)	1.5 mL
	1 M DTT	75 μL
	Zymolyase buffer	600 μL
	10% SDS (final concentration at 0.05%)	11 μL
Lysis buffer (no DTT)² (950 μL)	H₂O	500 μL
	20% Ficoll PM-100	300 μL
	Sarkosyl	10 μL
	0.5 M EDTA	40 μL
	2 M Tris pH 7.5	100 μL
Zymolyase buffer³⁷ (CSH protocols Zymolyase Buffer) (100 mL)	2 M Sorbitol	50 mL
	1 M K₂HPO₄	4.2 mL
	1 M KH₂PO₄	0.8 mL
	EDTA (0.5 M, pH 8)	1 mL
	H₂O	44 mL

Downstream breakage of oil droplets followed by reverse transcription, exonuclease I treatment, and PCR for cDNA amplification were carried out as per the Drop-Seq protocol². cDNA was then tagmented using the NEBNext Ultra II DNA Library Prep with Sample Purification Beads kit (NEB #E7103S).
Sequencing was done using the Illumina HiSeq2500 platform with 2 × 100 read pairs.

Urbonaite G., Lee J.T., Liu P., Parada G.E., Hemberg M, & Acar M. (2021). A yeast-optimized single-cell transcriptomics platform elucidates how mycophenolic acid and guanine alter global mRNA levels. Communications Biology, 4, 822.

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Micrococcal Nuclease-based Chromatin Digestion

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Micrococcal nuclease (MNase) digestion was done essentially as described previously (29 (link)). Briefly, yeast cells were grown in SD medium at 30°C to late log phase. Half of the culture was irradiated with 120 J/m² of UV followed by incubation at 30°C for 1 h. Cells from 45 ml of the irradiated and unirradiated samples were treated with 50 units of Zymolyase (Zymo Research) in 5 ml Zymolyase buffer (50 mM Tris, pH 7.8, 1 M sorbitol, 5 mM β-ME, 0.5 mM PMSF) at 30°C for 40 min. The resulting spheroplasts from each sample were then suspended in 2 ml of spheroplast digestion buffer (50 mM Tris, pH 7.8, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl₂, 1 mM CaCl₂, 1 mM β-ME, 0.5 mM PMSF and 0.075% v/v NP-40), divided into 300-μl aliquots and digested with varying concentrations of MNase for 10 min at 37°C. The reactions were terminated by mixing with 60 μl of stop solution (6% SDS, 250 mM EDTA) and immediately incubated at 65°C for 3 h. The genomic DNA was isolated from the aliquots and fractionated on 1.2% agarose gels.

Selvam K., Rahman S.A, & Li S. (2019). Histone H4 H75E mutation attenuates global genomic and Rad26-independent transcription-coupled nucleotide excision repair. Nucleic Acids Research, 47(14), 7392-7401.

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Cell Wall Viscosity Reduction Imaging

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To decrease the viscosity of the cell wall, we utilized Zymolyase, which contains β-1,3 glucanase, to hydrolyze the glucan linkages that strengthen the wall. Zymolyase (Zymo Research, 1 μl (2 units) per 100 μl of cells) was added to cells exposed to alpha-factor for 1.5 hours. Cells were treated additionally with concanavalin A to immobilize them during the imaging process. The cells were imaged on slides for 7 minutes after being exposed to Zymolyase for 3 minutes. DIC images were acquired every 3 seconds. Data from 5 samples for each condition was averaged and, for each sample, 15 cells or more were analyzed. Image analysis was manually performed using ImageJ.

Banavar S.P., Gomez C., Trogdon M., Petzold L.R., Yi T.M, & Campàs O. (2018). Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis. PLoS Computational Biology, 14(1), e1005940.

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Sporulation and tetrad dissection of yeast strains

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All strains were derivatives of a modified version (Rad5⁺) of S. cerevisiae strain W303 (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15, RAD5⁺). Supplementary file 5 lists each strain’s genotype. The ancestors of WT and ctf4Δ strains were obtained by sporulating a CTF4/ctf4Δ heterozygous diploid. This was done to minimize the selection acting on the ancestor strains before the beginning of the experiment. Diploid stains were grown on YPD, transferred to sporulation plates (sodium acetate 0.82%, potassium chloride 0.19%, sodium chloride 0.12%, magnesium sulfate 0.035%) and incubated for four days at 25°C. Tetrads were re-suspended in water containing zymolyase (Zymo research, RRID:SCR_008968, Irvine, CA, US, 0.025 u/μl), incubated at 37°C for 45 s, and dissected on a YPD plate using a Nikon eclipse E400 microscope equipped with a TDM micro-manipulator. Spores were allowed to grow into visible colonies and genotyped by presence of genetic markers and PCR.

Fumasoni M, & Murray A.W. (2020). The evolutionary plasticity of chromosome metabolism allows adaptation to constitutive DNA replication stress. eLife, 9, e51963.

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Yeast Spheroplast Preparation and Protein Synthesis

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Yeast spheroplasts were prepared from a 50-ml culture grown to an OD600 of 0.8 by adding 350 U of zymolyase (Zymo Research) and incubating at 30 °C for 25–30 min as previously described^{22 (link)}. Spheroplasts were combined with synthetic sdRNA (10–100 pmol) or 100 pmol control RNA oligomers (scr-sdR128 5′-CUUGAGAUGAUUGCUAUGAUAC-3′, scr-ranc18 5′-AAGUGAAGAAGGAAGAAA-3′ or spike-in RNA 5′-AUAGGCCAUAAGGAGUCUCGGUACGUCUUGUAUG-3′) and electroporated. For controls, translation was inhibited by adding 7.5 μg/μl cycloheximide to the spheroplasts. Electroporated spheroplasts were incubated at 30°C with 1 μl ³⁵S-methionine (1 000 Ci/mmol, 10 mCi/ml) for 1 h. Labelled proteins were precipitated in TCA, recovered on Whatman glass fibre GF/C filters and subjected to scintillation counting. Metabolic labelling measurements were performed at least in triplicate, and standard error (SE) was calculated. Statistical significance was determined using t-test.

Mleczko A.M., Machtel P., Walkowiak M., Wasilewska A., Pietras P.J, & Bąkowska-Żywicka K. (2019). Levels of sdRNAs in cytoplasm and their association with ribosomes are dependent upon stress conditions but independent from snoRNA expression. Scientific Reports, 9, 18397.

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PLATE-seq protocol for yeast cell lysis

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Each colony was picked 6 times into 96-well plates containing 15 μL of 2.5 mg/mL Zymolyase (catalog no. E1004; Zymo Research) and incubated for 45 min at 37 °C followed by 10 min at 95 °C to prepare yeast cell lysate used as PLATE-seq DNA template. PLATE-seq was carried out as previously described 26 . In brief, plasmid(s) from individual wells of 96-well plates were PCR amplified using a plasmid-specific forward primer and a reverse primer consisting of a well-position-specific barcode and TruSeq 3′ sequencing adapter. Amplicons derived from the same 96-well plate were pooled and purified using QIAquick PCR Purification Kit (ca talog no.

Zhou Y., Liu Y., Gupta S., Paramo M.I., Hou Y., Mao C., Luo Y., Judd J., Wierbowski S.D., Bertolotti M., Nerkar M., Jehi L., Drayman N., Nicolaescu V., Gula H., Tay S., Prescott M., Lis J.T., Feschotte C., Erzurum S.C., Cheng F., & Yu H. (2022). A comprehensive SARS-CoV-2-human protein-protein interactome network reveals novel pathobiology and host-targeting therapies for COVID-19.

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Optimized DNA Isolation from Microbial Cultures

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For DNA isolation 10 ml of culture at mid-log phase (~ 3×10⁷ cells ml⁻¹) was collected and frozen. The protocol of Karas et al. (25 (link)) was utilized with some modifications. Briefly, for each reaction 235 μl Qiagen P1 solution was combined with 5 μl lysozyme (25 mg/ml) (Sigma-Aldrich), 2.5 μl macerozyme (100 mg/ml)(Yakult Pharmaceuticals), 2.5 μl zymolyase (10 mg/ml) (Zymo Research), and 5 μl cellulase (100mg/ml)(Yakult Pharmaceuticals) and used to resuspend the cell pellets. The resuspended cell culture was incubated at 37° C for 30 min. After addition of the P2 solution, and S3 solution (Qiagen), the cell debris was pelleted at 14000 × G for 10 min. Supernatant was aspirated and combined with an equal volume of isopropanol, mixed and centrifuged at 14,000 × G at 4° C for 10 min. After decanting the pellet was washed with 750 μl 70% ethanol, centrifuged, decanted, and dried. The isolated DNA was resuspended in Tris-EDTA buffer (TE) and re-isolated by phenol:chloroform extraction. The final DNA was resuspended in 40 μl TE, and the concentration determined using a NanoDrop Lite (Thermo Scientific).

Poliner E., Takeuchi T., Du Z.Y., Benning C, & Farré E.M. (2019). Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. The Plant journal : for cell and molecular biology, 99(1), 112-127.

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Antibody Library Sequencing Workflow

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Genome extraction was performed on yeast cultures of libraries and sorted rounds underwent genome extraction using a commercial kit (Promega, cat. no. A1120) with zymolyase (Zymo Research, cat. no. E1004). 100 ng genomic template was used to amplify the heavy and light chains separately or as one amplicon for short or long-read sequencing, respectively. For amplification of heavy chain genes only, primers JG.VHVLK.F and JG.VH.R were used. For amplification of light chain genes only, primers JG.VL.F and JG.VHVK.R or JG.VHVL.R were used for kappa and lambda vectors, respectively. For amplification of paired genes, primers JG.VHVLK.F and JG.VHVK.R or JG.VHVL.R were used. Amplicons were column purified and deep sequenced with an iSeq. In parallel, we obtained ~1.8 kb sequences spanning the entire VH and VL using MinION nanopore sequencing (Oxford Nanopore Technologies Ltd., MinION R10.3).

Goike J., Hsieh C.L., Horton A., Gardner E.C., Bartzoka F., Wang N., Javanmardi K., Herbert A., Abbassi S., Renberg R., Johanson M.J., Cardona J.A., Segall-Shapiro T., Zhou L., Nissly R.H., Gontu A., Byrom M., Maranhao A.C., Battenhouse A.M., Gejji V., Soto-Sierra L., Foster E.R., Woodard S.L., Nikolov Z.L., Lavinder J., Voss W.N., Annapareddy A., Ippolito G.C., Ellington A.D., Marcotte E.M., Finkelstein I.J., Hughes R.A., Musser J.M., Kuchipudi S.V., Kapur V., Georgiou G., Dye J.M., Boutz D.R., McLellan J.S, & Gollihar J.D. (2021). Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality. bioRxiv.

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Immunostaining and Western Blot Analysis

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For immunostaining, cells expressing Gcn3-HA were fixed via treatment with 3.7% formaldehyde (EMS, Hatfield, USA) for at least 30 min followed by 45 min incubation in spheroplasting buffer [100 mM phosphate buffer pH 7.5, 5 mM EDTA, 1.2 M Sorbitol (Sigma-Aldrich, Steinheim, Germany), Zymolyase (Zymo Research, USA)] at 30˚C with mild agitation. Spheroplasts were permeabilized with 1% triton X-100 (Serva, Heidelberg, Germany), washed and then incubated with α-HA primary antibody from mouse (1:2000; Covance, USA) and Alexa Fluor 488 F(ab′)₂ fragment of rabbit anti-mouse IgG (H+L) (1:2000; Invitrogen, A21204). GFP-tagged proteins were detected in lysates via western blot analysis using an α-GFP primary antibody (1:2000; Roche, Mannheim, Germany) and a secondary α-mouse antibody (1:5000; Sigma-Aldrich, St Louis, USA). The protein levels of PGK were determined as internal loading control with a α-PGK primary antibody from mouse (1:5000; Invitrogen, Camarillo, USA) and a secondary α-mouse antibody (1:5000; Sigma-Aldrich, St Louis, USA).

Nüske E., Marini G., Richter D., Leng W., Bogdanova A., Franzmann T.M., Pigino G, & Alberti S. (2020). Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells. Biology Open, 9(7), bio046391.

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Immunofluorescence Imaging of ATP7B in Yeast

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Yeast cells were cultured to mid-log phase in the ironlimited medium. Harvested yeast cells were fixed in 5 ml of 50 mM KPO 4 (pH 6.5), 1 mM MgCl 2 and 4% formaldehyde for 2 h. After fixation, the cells were washed two times in 5 ml of PM buffer (100 mM KPO 4 pH 7.5, 1 mM MgCl 2 ) and followed by resuspension in PMST buffer (100 mM KPO 4 pH 7.5, 1 mM MgCl 2 , 1 M sorbitol, 0.1% Triton X-100) to a final OD600 of 10. 100 ll yeast cells were incubated for 20 min in 0.6 ll of b-mercaptoethanol and 1 mg/ml zymolyase (Zymo Research). The spheroplasted cells were washed with PMST buffer and attached to polylysine-coated coverslips. Adherent cells were blocked in PMST-BSA buffer (0.5% BSA in PMST buffer) for 30 min. Next, the adherent cells were incubated overnight at 4 °C with primary antibody (1:500 rabbit monoclonal ATP7B antibody, Abcam) diluted in PMST-BSA buffer. After incubation, the cells were washed three times with PMST-BSA buffer and incubated with secondary antibody (1:1000 anti-rabbit Alexa 488, Abcam) for 3 h at room temperature, and with 0.4 mg/ml DAPI (staining nuclei) for 5 min. Cells were mounted in Vectashield mounting medium (Vector Laboratories). Images were acquired using a Leica DM 2000 inverted microscope and processed with the Leica application suite (LAS-AF lite) software.

Shanmugavel K.P., Kumar R., Li Y, & Wittung-Stafshede P. (2019). Wilson disease missense mutations in ATP7B affect metal-binding domain structural dynamics. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 32(6).

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