recombinant shrimp alkaline phosphatase  (New England Biolabs)


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    Structured Review

    New England Biolabs recombinant shrimp alkaline phosphatase
    The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with <t>shrimp</t> <t>alkaline</t> <t>phosphatase</t> for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a <t>recombinant</t> TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.
    Recombinant Shrimp Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant shrimp alkaline phosphatase/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant shrimp alkaline phosphatase - by Bioz Stars, 2022-05
    97/100 stars

    Images

    1) Product Images from "Promoter-proximal elongation regulates transcription in archaea"

    Article Title: Promoter-proximal elongation regulates transcription in archaea

    Journal: Nature Communications

    doi: 10.1038/s41467-021-25669-2

    The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with shrimp alkaline phosphatase for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a recombinant TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.
    Figure Legend Snippet: The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with shrimp alkaline phosphatase for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a recombinant TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.

    Techniques Used: CRISPR, In Vitro, Plasmid Preparation, Recombinant, Variant Assay, Generated, Purification, Affinity Purification, In Vivo

    2) Product Images from "Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation"

    Article Title: Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation

    Journal: bioRxiv

    doi: 10.1101/407031

    a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see Figure 1c ). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler mass spectrometry 34 . We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy34. Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the damC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´ → 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The damC sequencing library preparation protocol includes UMIs allowing to filter ∼15% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID30 at the same sequencing depth. f) Median DamC enrichment at 100 viewpoints with highest enrichment as a function of 4-OHT concentration. Significant amounts of damC enrichment can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5-10 nM 4-OHT in our experimental system.
    Figure Legend Snippet: a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see Figure 1c ). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler mass spectrometry 34 . We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy34. Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the damC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´ → 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The damC sequencing library preparation protocol includes UMIs allowing to filter ∼15% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID30 at the same sequencing depth. f) Median DamC enrichment at 100 viewpoints with highest enrichment as a function of 4-OHT concentration. Significant amounts of damC enrichment can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5-10 nM 4-OHT in our experimental system.

    Techniques Used: Concentration Assay, Binding Assay, Mass Spectrometry, Staining, Expressing, Ligation, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, Methylation, Genome Wide

    3) Product Images from "Innate Biomineralization"

    Article Title: Innate Biomineralization

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21144820

    Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.
    Figure Legend Snippet: Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.

    Techniques Used: Staining, Cell Culture, Cell Differentiation, Titration

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    New England Biolabs recombinant shrimp alkaline phosphatase
    The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with <t>shrimp</t> <t>alkaline</t> <t>phosphatase</t> for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a <t>recombinant</t> TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.
    Recombinant Shrimp Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant shrimp alkaline phosphatase/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant shrimp alkaline phosphatase - by Bioz Stars, 2022-05
    97/100 stars
      Buy from Supplier

    99
    New England Biolabs shrimp alkaline phosphatase
    The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with <t>shrimp</t> <t>alkaline</t> <t>phosphatase</t> for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a <t>recombinant</t> TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.
    Shrimp Alkaline Phosphatase, supplied by New England Biolabs, 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/shrimp alkaline phosphatase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase - by Bioz Stars, 2022-05
    99/100 stars
      Buy from Supplier

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    The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with shrimp alkaline phosphatase for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a recombinant TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.

    Journal: Nature Communications

    Article Title: Promoter-proximal elongation regulates transcription in archaea

    doi: 10.1038/s41467-021-25669-2

    Figure Lengend Snippet: The CRISPR C promoter shows early pausing in vitro. a Schematic overview of the cell lysate-based synchronised in vitro transcription system for S. solfataricus . Cell lysates were treated with shrimp alkaline phosphatase for NTP degradation before heat inactivation of the phosphatase. Linearised plasmid DNA containing a S. solfataricus promoter were added to allow PIC formation on the templates with inherent initiation factor TFB. Simultaneously with the addition of ribonucleotides to allow the PICs to initiate a single round of transcription, we added an excess of a recombinant TFB variant termed TFBc that blocks subsequent rounds of PIC formation. The generated transcripts were purified by affinity purification using immobilised 25 nt antisense oligonucleotides. b Synchronised in vitro transcription assay with two promoters showing high TEC escape ( thsB and rps8e ) and two promoters showing low TEC escape in vivo ( dhg-1 and CRISPR C ). Samples were withdrawn 15 s, 30 s and 45 s after simultaneous addition of 50 µM rNTPs including [α- 32 P]-UTP and TFBc. Purified radiolabelled transcripts were resolved on a denaturing polyacrylamide gel. The position of run-off transcripts and transcripts resulting from pausing in the promoter-proximal region is indicated. A representative experiment of three technical replicates is shown.

    Article Snippet: Before using the cell lysates in in vitro transcription reactions, nucleotides were degraded by treating the lysate with 100 unit/ml recombinant shrimp alkaline phosphatase (New England Biolabs) for 20 min at 37 °C followed by 5 min at 65 °C to inactivate the phosphatase.

    Techniques: CRISPR, In Vitro, Plasmid Preparation, Recombinant, Variant Assay, Generated, Purification, Affinity Purification, In Vivo

    a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see Figure 1c ). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler mass spectrometry 34 . We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy34. Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the damC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´ → 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The damC sequencing library preparation protocol includes UMIs allowing to filter ∼15% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID30 at the same sequencing depth. f) Median DamC enrichment at 100 viewpoints with highest enrichment as a function of 4-OHT concentration. Significant amounts of damC enrichment can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5-10 nM 4-OHT in our experimental system.

    Journal: bioRxiv

    Article Title: Modeling of DNA methylation in cis reveals principles of chromatin folding in vivo in the absence of crosslinking and ligation

    doi: 10.1101/407031

    Figure Lengend Snippet: a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see Figure 1c ). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler mass spectrometry 34 . We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy34. Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the damC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´ → 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The damC sequencing library preparation protocol includes UMIs allowing to filter ∼15% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID30 at the same sequencing depth. f) Median DamC enrichment at 100 viewpoints with highest enrichment as a function of 4-OHT concentration. Significant amounts of damC enrichment can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5-10 nM 4-OHT in our experimental system.

    Article Snippet: Genomic DNA (100ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step.

    Techniques: Concentration Assay, Binding Assay, Mass Spectrometry, Staining, Expressing, Ligation, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, Methylation, Genome Wide

    Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.

    Journal: International Journal of Molecular Sciences

    Article Title: Innate Biomineralization

    doi: 10.3390/ijms21144820

    Figure Lengend Snippet: Calcium ion (Ca 2+ ), a phosphoester salt, and alkaline phosphatase (ALP) are essential for promoting biomineralization in MG-63 and Saos-2 cell lines and human blood mononuclear cells (MNCs). ( A ) Alizarin Red S assays (ARS) show mineralization within 7 days, visualized as the intensity of red staining, of Saos-2 cells in MEMα/10% FBS supplemented only with βGP or the combination of βGP/ascorbic acid (Vit. C)/dexamethasone (Dex); MG-63 cell line was inactive under similar conditions. No mineralization was observed in either cell line cultured with MEMα/10% FBS supplemented with Vit. C or Dex. In MEMα/10% FBS supplemented with βGP and ALPL, calf intestinal ALP (CIP), or shrimp ALP (SAP), MG-63 and Saos-2 cell lines were mineralized. ( B ) Similar results were iterated when αGP was used instead of βGP. ( C ) Compared to βGP and phosphoenolpyruvate monosodium (PEP), αGP was the most efficient phosphoester salt elicited biomineralization. Pamidronate (Pamidn) and glycerophosphoric acid (NSC9231) did not elicit the reaction. ( D ) Human MNCs also have an innate ability of mineralization in 7 days without the induction of cellular differentiation (top row indicates initial cell counts per well in a 6-well plate). ( E – G ) Titration assays indicated that biomineralization depended on the doses of αGP, CIP, and Ca 2+ . ( H ) In a 48-well plate, human blood MNCs were seeded (10 5 /well) and exposed to MEMα/10% FBS, the medium supplemented with αGP, with αGP and CIP, or with CIP for 7 days (the media were changed on day 4). On day 7, ARS indicates that biomineralization occurred in MNCs exposed to MEMα/10% FBS supplemented with αGP and CIP in the uncoated wells (top row) and the wells coated with Collagen Type I, rat tail (middle row). Biomineralization did not occur if αGP or CIP was missing. None of the cell-less wells coated with Collagen Type I was positive for HAP by ARS (bottom row). Each of the tests was in duplicates.

    Article Snippet: CIP (≥10,000 U/mL), shrimp hepatopancreas ALP (≥1000 unit/mL), and p-nitrophenyl phosphate kits were purchased from New England BioLabs (Ipswich, MA, USA).

    Techniques: Staining, Cell Culture, Cell Differentiation, Titration