isothermal reaction buffer  (Thermo Fisher)


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  • 99
    Name:
    Reaction Buffer
    Description:
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    Catalog Number:
    b43
    Price:
    None
    Applications:
    In Vitro Transcription|One-Step qRT-PCR|PCR & Real-Time PCR|RT-PCR|Real Time PCR (qPCR)|Reverse Transcription|Two-Step RT-PCR|Gene Expression Analysis & Genotyping
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher isothermal reaction buffer
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    https://www.bioz.com/result/isothermal reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    isothermal reaction buffer - by Bioz Stars, 2020-09
    99/100 stars

    Images

    Related Articles

    Conjugation Assay:

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Amplification:

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    Labeling:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Purification:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    SYBR Green Assay:

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

    Incubation:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Article Title: Rapid cleavage of RNA by RNase E in the absence of 5? monophosphate stimulation
    Article Snippet: .. Conjugation of 5′-biotinylated oligonucleotides to streptavidin Increasing amounts of 5′-biotinylated LU13 (0.15, 0.3, 0.6 and 1.5 nmol) were incubated with streptavidin from Streptomyces avidinii (Sigma) (0.15 nmol) in 100 μl of RNase E reaction buffer ( ; ) containing 80 U of RNaseOUT (Invitrogen) at 30°C for 20 min. .. Samples of the reaction products were added to an equal volume of loading buffer [100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue] and analysed by native gel electrophoresis using 12% (w/v) 29:1 acrylamide : bis -acrylamide gels containing 150 mM Tris-HCl, pH 6.8 in the upper stacking gel and 375 mM Tris-HCl, pH 8.8 in the resolving gel and electrophoresis buffer containing 192 mM glycine and 25 mM Tris-HCl, pH 8.3.

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

    Article Title: Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats
    Article Snippet: .. Briefly, unfixed cryosections were incubated for 15 min in calpain reaction buffer (CRB; 25 mM HEPES, 65 mM KCl, 2 mM MgCl2 , 1,5 mM CaCl2 , 2 mM DTT) and then sections were incubated at 35°C for 1 h in the dark in 2 µM fluorescent calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl- L-methionine amide (CMAC, t-BOC-Leu-Met; Molecular Probes, Inc. Eugene, USA). .. Fluorescence was generated by calpain-dependent cleavage of t-Boc-Leu-Met-CMAC.

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    Polymerase Chain Reaction:

    Article Title: Expression of c-Met and Heparan-Sulfate Proteoglycan Forms of CD44 in Colorectal Cancer
    Article Snippet: .. PCR was performed with 1.5 U Taq DNA Polymerase (Gibco BRL/Life Technologies), 200 μmol/L dNTPs (Pharmacia Biotech, Uppsala, Sweden), and 1.5 mmol/L MgCl2 (2 mmol/L for GAPDH) in 1× PCR Buffer (both Gibco BRL/Life Technologies). ..

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. Seventeen microliters of the preamplification mix (10 µL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher), and 2 µL of 10× pooled primer mix in nuclease-free water) was added to 3 µL of the RT products.

    Staining:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Recombinant:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    SDS Page:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Molecular Weight:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

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    Thermo Fisher isothermal reaction buffer
    Isothermal Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isothermal reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    isothermal reaction buffer - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    84
    Thermo Fisher mto2 tcd
    Mutations in the <t>Mto2</t> NND and <t>TCD</t> impair Mto1/2 function in vivo . (A) Microtubule organization and Mto1[bonsai]-GFP puncta formation in wild-type cells ( mto2+) and in the mto2 mutants indicated. Quantification of microtubule organization is shown in Suppl. Figure 6 . Scale bar, 10 µm. (B) Western blots for Mto1, Mto2, and γ -tubulin (Gtb1), showing coimmunoprecipitation with Mto1[bonsai]-GFP in wild-type cells and in the mto2 mutants indicated. Left-most lane is from cells with untagged full-length Mto1. (C, D) Quantification of coimmunoprecipitation of Mto2 (C) and Gtb1 (D) with Mto1[bonsai]-GFP, from blots in B.
    Mto2 Tcd, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mto2 tcd/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mto2 tcd - by Bioz Stars, 2020-09
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    Image Search Results


    Mutations in the Mto2 NND and TCD impair Mto1/2 function in vivo . (A) Microtubule organization and Mto1[bonsai]-GFP puncta formation in wild-type cells ( mto2+) and in the mto2 mutants indicated. Quantification of microtubule organization is shown in Suppl. Figure 6 . Scale bar, 10 µm. (B) Western blots for Mto1, Mto2, and γ -tubulin (Gtb1), showing coimmunoprecipitation with Mto1[bonsai]-GFP in wild-type cells and in the mto2 mutants indicated. Left-most lane is from cells with untagged full-length Mto1. (C, D) Quantification of coimmunoprecipitation of Mto2 (C) and Gtb1 (D) with Mto1[bonsai]-GFP, from blots in B.

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: Mutations in the Mto2 NND and TCD impair Mto1/2 function in vivo . (A) Microtubule organization and Mto1[bonsai]-GFP puncta formation in wild-type cells ( mto2+) and in the mto2 mutants indicated. Quantification of microtubule organization is shown in Suppl. Figure 6 . Scale bar, 10 µm. (B) Western blots for Mto1, Mto2, and γ -tubulin (Gtb1), showing coimmunoprecipitation with Mto1[bonsai]-GFP in wild-type cells and in the mto2 mutants indicated. Left-most lane is from cells with untagged full-length Mto1. (C, D) Quantification of coimmunoprecipitation of Mto2 (C) and Gtb1 (D) with Mto1[bonsai]-GFP, from blots in B.

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: In Vivo, Western Blot

    Alternative model for Mto1/2 complex architecture. In this variant of the model in Figure 7 , Mto2 C-terminus interacts with a different NND-NND interface from that shown in Figure 7 (e.g. the C-terminus of Mto2 n interacts with the NND-NND interface between Mto2 n+1 and Mto2 n+2 , rather than between Mto2 n and Mto2 n-1 . For this model to be plausible, the constraints on NND and TCD orientation that prevent closed bivalent dimer formation ( Suppl. Figure 5 ) would likely be conferred by the linker sequence between the NND and the TCD rather than by the C-terminus interacting with the NND (see Figure 7 ). Although this linker is predicted to be intrinsically disordered, it might adopt a specific structure in the presence of the NND and/or TCD.

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: Alternative model for Mto1/2 complex architecture. In this variant of the model in Figure 7 , Mto2 C-terminus interacts with a different NND-NND interface from that shown in Figure 7 (e.g. the C-terminus of Mto2 n interacts with the NND-NND interface between Mto2 n+1 and Mto2 n+2 , rather than between Mto2 n and Mto2 n-1 . For this model to be plausible, the constraints on NND and TCD orientation that prevent closed bivalent dimer formation ( Suppl. Figure 5 ) would likely be conferred by the linker sequence between the NND and the TCD rather than by the C-terminus interacting with the NND (see Figure 7 ). Although this linker is predicted to be intrinsically disordered, it might adopt a specific structure in the presence of the NND and/or TCD.

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: Variant Assay, Sequencing

    The Mto2 Near-N-terminal Domain (NND) and Twin-Cysteine Domain (TCD) each form homodimers. (A) Size-exclusion chromatography with multi-angle laser-light scattering (SEC-MALS) analysis of the NND. Sample was injected at ∼5 mg/mL. Schematic of predicted alpha-helices indicates non-coiled-coil ( α H) and coiled-coil (CC) regions. In the tail following the protein peak, gradual decrease in molecular mass (to 50% of peak value) indicates dissociation of dimers into monomers. See also Suppl. Figure 2B,D . (B) Isothermal titration calorimetry analysis of NND homodimer dissociation. (C) SDS-PAGE of wild-type NND and mutants NND-N123C and NND-R147C, under reducing and non-reducing conditions (with and without β -mercaptoethanol; β -ME). Dimer bands indicate intermolecular disulfide bond formation under non-reducing conditions, suggesting that CC region forms a parallel homodimer. (D) SEC-MALS analysis of the TCD. Sample was injected at ∼10 mg/mL. Schematic indicates predicted alpha helices (H1, H2, H3). In the tail following the protein peak, molecular mass remains constant. See also Suppl. Figure 2C,E .

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: The Mto2 Near-N-terminal Domain (NND) and Twin-Cysteine Domain (TCD) each form homodimers. (A) Size-exclusion chromatography with multi-angle laser-light scattering (SEC-MALS) analysis of the NND. Sample was injected at ∼5 mg/mL. Schematic of predicted alpha-helices indicates non-coiled-coil ( α H) and coiled-coil (CC) regions. In the tail following the protein peak, gradual decrease in molecular mass (to 50% of peak value) indicates dissociation of dimers into monomers. See also Suppl. Figure 2B,D . (B) Isothermal titration calorimetry analysis of NND homodimer dissociation. (C) SDS-PAGE of wild-type NND and mutants NND-N123C and NND-R147C, under reducing and non-reducing conditions (with and without β -mercaptoethanol; β -ME). Dimer bands indicate intermolecular disulfide bond formation under non-reducing conditions, suggesting that CC region forms a parallel homodimer. (D) SEC-MALS analysis of the TCD. Sample was injected at ∼10 mg/mL. Schematic indicates predicted alpha helices (H1, H2, H3). In the tail following the protein peak, molecular mass remains constant. See also Suppl. Figure 2C,E .

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: Size-exclusion Chromatography, Injection, Isothermal Titration Calorimetry, SDS Page

    Mutations affecting NND dimerization. (A) Helical-wheel plot of the predicted coiled-coil region within the Mto2 NND (residues 123-148). Dashed lines indicate predicted salt bridges. Residues mutated in the 134A136A, 134AAA, and 129AAA mutants are indicated below (the 129AAA mutation is relevant to Figure 6 and Suppl. Figure 6 ). (B,C) SEC-MALS analysis of purified mutant NND proteins NND-134A136A (B) and NND-134AAA (C). Samples were injected at 5.4 mg/mL (B) and 1.3 mg/mL (C). Unlike wild-type NND, which forms homodimers ( Figure 2 , Suppl. Figure 2 ), both mutant proteins are monomeric. (D,E) SEC-MALS analysis of full-length Mto2 containing the 134A136A (D) and 134AAA (E) mutations. Samples were injected at 1.4 mg/mL (D) and 1.3 mg/mL (E).Unlike wild-type Mto2, which forms higher-order multimers ( Figure 1 ; Suppl. Figure 1 ), both Mto2-134A136A and Mto2-134AAA are dimeric. Different Y-axis units (RI signal, dRI) are used for indicating protein peaks in D and E because experiments were performed on different instruments (see Methods). (F) Additional controls for experiments showing effects of NND and TCD overexpression in mto1Δ cells, as in Figure 4C . In mto1Δ cells expressing mto2-GFP but lacking transgenes, Mto2-GFP puncta are unaffected by the presence vs. the absence of thiamine. In mto1Δ cells lacking Mto2-GFP ( mto2+ untagged), neither fluorescent puncta nor cytoplasmic fluorescence is observed. Images were processed identically to those shown in Figure 4C . Scale bar, 10 µm. Note log 10 scales for molecular mass in B-E.

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: Mutations affecting NND dimerization. (A) Helical-wheel plot of the predicted coiled-coil region within the Mto2 NND (residues 123-148). Dashed lines indicate predicted salt bridges. Residues mutated in the 134A136A, 134AAA, and 129AAA mutants are indicated below (the 129AAA mutation is relevant to Figure 6 and Suppl. Figure 6 ). (B,C) SEC-MALS analysis of purified mutant NND proteins NND-134A136A (B) and NND-134AAA (C). Samples were injected at 5.4 mg/mL (B) and 1.3 mg/mL (C). Unlike wild-type NND, which forms homodimers ( Figure 2 , Suppl. Figure 2 ), both mutant proteins are monomeric. (D,E) SEC-MALS analysis of full-length Mto2 containing the 134A136A (D) and 134AAA (E) mutations. Samples were injected at 1.4 mg/mL (D) and 1.3 mg/mL (E).Unlike wild-type Mto2, which forms higher-order multimers ( Figure 1 ; Suppl. Figure 1 ), both Mto2-134A136A and Mto2-134AAA are dimeric. Different Y-axis units (RI signal, dRI) are used for indicating protein peaks in D and E because experiments were performed on different instruments (see Methods). (F) Additional controls for experiments showing effects of NND and TCD overexpression in mto1Δ cells, as in Figure 4C . In mto1Δ cells expressing mto2-GFP but lacking transgenes, Mto2-GFP puncta are unaffected by the presence vs. the absence of thiamine. In mto1Δ cells lacking Mto2-GFP ( mto2+ untagged), neither fluorescent puncta nor cytoplasmic fluorescence is observed. Images were processed identically to those shown in Figure 4C . Scale bar, 10 µm. Note log 10 scales for molecular mass in B-E.

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: Mutagenesis, Purification, Injection, Over Expression, Expressing, Fluorescence

    X-ray crystal structure of the Mto2 Twin-Cysteine Domain (TCD). (A) Ribbon diagram of the TCD dimer. The two monomer chains (TCDa and TCDb) are in different shades of blue. Rotated view shows proximity of the two three-helix bundles. See also Suppl. Figure 3 . (B) Surface-charge representation of the TCD monomer. Left panel includes ribbon diagram of second monomer, to show orientation of the dimer relative to A. Note absence of charged residues at the hydrophobic dimer interface (right panel, white surface).

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: X-ray crystal structure of the Mto2 Twin-Cysteine Domain (TCD). (A) Ribbon diagram of the TCD dimer. The two monomer chains (TCDa and TCDb) are in different shades of blue. Rotated view shows proximity of the two three-helix bundles. See also Suppl. Figure 3 . (B) Surface-charge representation of the TCD monomer. Left panel includes ribbon diagram of second monomer, to show orientation of the dimer relative to A. Note absence of charged residues at the hydrophobic dimer interface (right panel, white surface).

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques:

    Mto2-Mto2 and Mto2-Mto1 interactions identified by crosslinking mass spectrometry. (A) Mto2-Mto2 interactions identified by EDC crosslinking of Mto2 alone. Light-blue shading indicates links between C terminus and NND, and links from both of these regions to TCD. (B) In vivo puncta formation for wild-type Mto2-mECitrine (Mto2-mEC) and Mto2 C-terminal truncations, in mto1Δ strain background. No puncta are seen with truncation Mto2[1-319]-mEC. (C) Mto1-Mto2, Mto2-Mto2, and Mto1-Mto1 interactions identified by EDC crosslinking of Mto1/2[bonsai] complex. For clarity, for Mto1-Mto1 crosslinks, only unambiguously intermolecular crosslinks are shown. Additional Mto1-Mto1 crosslinks from the same experiment are shown in Suppl. Figure 5C . (D) Copurification of wild-type Mto2 and Mto2 mutants with GST-Mto1[bonsai]. Upper panels show SDS-PAGE Coomassie Blue stain. Lower panels show anti-Mto2 western blot, with quantification. In A and C, red and green links are intermolecular; purple links could in principle be either intra- or intermolecular (see Discussion). Within each protein, potentially crosslinkable residues are indicated by green and red vertical lines, representing nucleophilic and carboxylic acid groups, respectively.

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: Mto2-Mto2 and Mto2-Mto1 interactions identified by crosslinking mass spectrometry. (A) Mto2-Mto2 interactions identified by EDC crosslinking of Mto2 alone. Light-blue shading indicates links between C terminus and NND, and links from both of these regions to TCD. (B) In vivo puncta formation for wild-type Mto2-mECitrine (Mto2-mEC) and Mto2 C-terminal truncations, in mto1Δ strain background. No puncta are seen with truncation Mto2[1-319]-mEC. (C) Mto1-Mto2, Mto2-Mto2, and Mto1-Mto1 interactions identified by EDC crosslinking of Mto1/2[bonsai] complex. For clarity, for Mto1-Mto1 crosslinks, only unambiguously intermolecular crosslinks are shown. Additional Mto1-Mto1 crosslinks from the same experiment are shown in Suppl. Figure 5C . (D) Copurification of wild-type Mto2 and Mto2 mutants with GST-Mto1[bonsai]. Upper panels show SDS-PAGE Coomassie Blue stain. Lower panels show anti-Mto2 western blot, with quantification. In A and C, red and green links are intermolecular; purple links could in principle be either intra- or intermolecular (see Discussion). Within each protein, potentially crosslinkable residues are indicated by green and red vertical lines, representing nucleophilic and carboxylic acid groups, respectively.

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: Mass Spectrometry, In Vivo, Copurification, SDS Page, Staining, Western Blot

    Mto1/2[bonsai] complex and Mto2 each form higher-older multimers in vitro . (A) Domain organization of Mto1[bonsai] (green) and Mto2 (blue). Residue numbering for Mto1[bonsai] corresponds to residues in full-length Mto1 (see Suppl. Figure 1A ). CM1 indicates Centrosomin Motif 1. NND and TCD indicate Near-N-terminal Domain and Twin-Cysteine Domain. (B) Size-exclusion chromatography (SEC) of purified Mto1/2[bonsai] complex at different dilutions. Undiluted sample was injected at ∼9 mg/mL. Arrowheads indicate multiple peaks due to dissociation. Corresponding Coomassie Blue (CB)-stained SDS-PAGE and anti-Mto1 and anti-Mto2 western blots are shown underneath. In CB-stained gels, upper band is Mto1[bonsai], and lower band is Mto2. (C, D) SEC of purified Mto1[bonsai] (C) and purified Mto2 (D) at different dilutions. Undiluted samples were injected at ∼1.5 mg/mL (Mto1[bonsai]) and ∼2.5 mg/ml (Mto2). SDS-PAGE and western blots are shown underneath. See also Suppl. Figure 1 .

    Journal: bioRxiv

    Article Title: Architecture of the Mto1/2 microtubule nucleation complex

    doi: 10.1101/754457

    Figure Lengend Snippet: Mto1/2[bonsai] complex and Mto2 each form higher-older multimers in vitro . (A) Domain organization of Mto1[bonsai] (green) and Mto2 (blue). Residue numbering for Mto1[bonsai] corresponds to residues in full-length Mto1 (see Suppl. Figure 1A ). CM1 indicates Centrosomin Motif 1. NND and TCD indicate Near-N-terminal Domain and Twin-Cysteine Domain. (B) Size-exclusion chromatography (SEC) of purified Mto1/2[bonsai] complex at different dilutions. Undiluted sample was injected at ∼9 mg/mL. Arrowheads indicate multiple peaks due to dissociation. Corresponding Coomassie Blue (CB)-stained SDS-PAGE and anti-Mto1 and anti-Mto2 western blots are shown underneath. In CB-stained gels, upper band is Mto1[bonsai], and lower band is Mto2. (C, D) SEC of purified Mto1[bonsai] (C) and purified Mto2 (D) at different dilutions. Undiluted samples were injected at ∼1.5 mg/mL (Mto1[bonsai]) and ∼2.5 mg/ml (Mto2). SDS-PAGE and western blots are shown underneath. See also Suppl. Figure 1 .

    Article Snippet: Thermal denaturation assayPurified Mto2 TCD (45 µL of 20 µM purified protein in GST-Tris200 Buffer) was mixed with 5 µL of 1x SYPRO Orange dye (Thermo Fisher Scientific) and subjected to a temperature (20-95°C) vs fluorescence scan on an iQ5 Real-Time PCR system (BioRad).

    Techniques: In Vitro, Size-exclusion Chromatography, Purification, Injection, Staining, SDS Page, Western Blot