Lysates and extracts were prepared as previously described48 (link). Immunoprecipitation of Spo11-oligo complexes was performed using 5 μg of mouse monoclonal anti-flag M2 antibody (Sigma). Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32P]dCTP and terminal deoxynucleotidyl transferase (TdT)48 (link). Twenty-five μl of reaction mixture was added to the beads, mixed, and incubated at 37°C for 1–2 hr. Spo11-oligo complexes were eluted by adding 25 μl of NUPAGE® loading buffer (diluted to 2× and supplemented with 83.3 mM dithiothreitol) (Invitrogen) and boiling for 5 min. End-labeled Spo11-oligo complexes were separated on a Novex® 4–12% gradient denaturing polyacrylamide gel (Invitrogen) then transferred onto PVDF membrane using the iBlot protocol (Invitrogen) and visualized by phosphorimager. Blots were probed with mouse monoclonal anti-flag M2 conjugated to horseradish peroxidase (Sigma). Chemiluminescent detection was performed according to the manufacturer's instructions (ECL+ or ECL Prime, Amersham). Protein quantity was estimated by separating 1 μl of extract on a Novex® 4–12% gradient denaturing polyacrylamide gel and staining with Coomassie Brilliant Blue.
Mouse monoclonal anti flag m2 conjugated to horseradish peroxidase
Manufactured by Merck Group
The mouse monoclonal anti-flag M2 conjugated to horseradish peroxidase is a laboratory reagent used for the detection and identification of proteins tagged with the FLAG epitope. It consists of a mouse-derived monoclonal antibody specific to the FLAG peptide sequence, conjugated to the enzyme horseradish peroxidase. This product can be used in various immunoassay techniques, such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti flag m2 antibody, by Merck Group (4 mentions)
Ecl prime, by Cytiva (3 mentions)
Novex 4 12 gradient denaturing polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Iblot protocol, by Thermo Fisher Scientific (2 mentions)
Nebuffer 4, by New England Biolabs (2 mentions)
Nupage loading buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
4 protocols using mouse monoclonal anti flag m2 conjugated to horseradish peroxidase
1
Spo11-oligo Complexes Immunoprecipitation
2
Isolation and Mapping of Spo11-Oligo Complexes
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Spo11-oligo complexes were extracted and detected as previously described (Supplemental Fig. S3A ; Thacker et al. 2014 (link)). Briefly, Spo11-oligo complexes were immunoprecipitated from whole-cell extracts by using mouse monoclonal anti-FLAG M2 antibody (Sigma). Precipitated Spo11-oligo complexes were end-labeled with [α-32P]dCTP in a terminal deoxynucleotidyl transferase reaction, resolved by SDS-PAGE, then transferred onto PVDF membrane and visualized by PhosphorImager. Blots were probed with mouse monoclonal anti-FLAG M2 conjugated to horseradish peroxidase (Sigma) and detected by chemiluminescent (ECL+ or ECL Prime, Amersham).
For Spo11-oligo mapping, sporulation cultures of different volumes (450 mL for homeologous strain; 300 mL for trisomic strain; 600 mL for all other strains) were harvested at desired time points after transferring to sporulation media. Maps were generated for this study in strains carrying SPO11-FLAG as described (Murakami et al. 2020 (link)) except for the trisomic strain carrying SPO11-PrA. Previously published wild-type maps (Thacker et al. 2014 (link); Mohibullah and Keeney 2017 (link)) were used as controls in this study.
For Spo11-oligo mapping, sporulation cultures of different volumes (450 mL for homeologous strain; 300 mL for trisomic strain; 600 mL for all other strains) were harvested at desired time points after transferring to sporulation media. Maps were generated for this study in strains carrying SPO11-FLAG as described (Murakami et al. 2020 (link)) except for the trisomic strain carrying SPO11-PrA. Previously published wild-type maps (Thacker et al. 2014 (link); Mohibullah and Keeney 2017 (link)) were used as controls in this study.
3
Spo11-oligo Complexes Immunoprecipitation
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Lysates and extracts were prepared as previously described48 (link). Immunoprecipitation of Spo11-oligo complexes was performed using 5 μg of mouse monoclonal anti-flag M2 antibody (Sigma). Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32P]dCTP and terminal deoxynucleotidyl transferase (TdT)48 (link). Twenty-five μl of reaction mixture was added to the beads, mixed, and incubated at 37°C for 1–2 hr. Spo11-oligo complexes were eluted by adding 25 μl of NUPAGE® loading buffer (diluted to 2× and supplemented with 83.3 mM dithiothreitol) (Invitrogen) and boiling for 5 min. End-labeled Spo11-oligo complexes were separated on a Novex® 4–12% gradient denaturing polyacrylamide gel (Invitrogen) then transferred onto PVDF membrane using the iBlot protocol (Invitrogen) and visualized by phosphorimager. Blots were probed with mouse monoclonal anti-flag M2 conjugated to horseradish peroxidase (Sigma). Chemiluminescent detection was performed according to the manufacturer's instructions (ECL+ or ECL Prime, Amersham). Protein quantity was estimated by separating 1 μl of extract on a Novex® 4–12% gradient denaturing polyacrylamide gel and staining with Coomassie Brilliant Blue.
4
Mapping Genome-wide Spo11 Oligonucleotides
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Spo11-oligo complexes were extracted and detected as previously described (Supplemental Fig. S3A) (Neale and Keeney 2009) . Briefly, Spo11-oligo complexes were immunoprecipitated from whole-cell extracts by using mouse monoclonal anti-Flag M2 antibody (Sigma). Precipitated Spo11-oligo complexes were end-labeled with [α-32P ] dCTP in a terminal deoxynucleotidyl transferase reaction, resolved by SDS-PAGE, then transferred onto PVDF membrane and visualized by phosphorimager. Blots were probed with mouse monoclonal anti-Flag M2 conjugated to horseradish peroxidase (Sigma) and detected by chemiluminescent (ECL+ or ECL Prime, Amersham).
For Spo11-oligo mapping, sporulation cultures of different volumes (450 ml for homeologous strain; 300 ml for trisomic strain; 600 ml for all other strains) were harvested at desired time points after transferring to sporulation media. Maps were generated for this study in strains carrying SPO11-Flag as described (Lam et al. 2017 ) except for the trisomic strain carrying SPO11-PrA. Previously published wild-type maps (Thacker et al. 2014; Mohibullah and Keeney 2017) were used as controls in this study. For the PCA and clustering analyses, we also included other wild-type and zip3∆ maps (Zhu and Keeney 2015; Mohibullah and Keeney 2017; Murakami et al. 2020) .
For Spo11-oligo mapping, sporulation cultures of different volumes (450 ml for homeologous strain; 300 ml for trisomic strain; 600 ml for all other strains) were harvested at desired time points after transferring to sporulation media. Maps were generated for this study in strains carrying SPO11-Flag as described (Lam et al. 2017 ) except for the trisomic strain carrying SPO11-PrA. Previously published wild-type maps (Thacker et al. 2014; Mohibullah and Keeney 2017) were used as controls in this study. For the PCA and clustering analyses, we also included other wild-type and zip3∆ maps (Zhu and Keeney 2015; Mohibullah and Keeney 2017; Murakami et al. 2020) .
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