t4 pnk  (Thermo Fisher)


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  • 99
    Name:
    T4 Polynucleotide Kinase 10 U µL
    Description:
    Thermo Scientific T4 Polynucleotide Kinase T4 PNK catalyzes the transfer of the gamma phosphate from ATP to the 5 OH group of single and double stranded DNAs and RNAs oligonucleotides or nucleoside 3 monophosphates forward reaction The reaction is reversible In the presence of ADP T4 Polynucleotide Kinase exhibits 5 phosphatase activity and catalyzes the exchange of phosphate groups between 5 P oligo polynucleotides and ATP exchange reaction The enzyme is also a 3 phosphatase Highlights • Active in Thermo Scientific restriction enzyme RT and T4 DNA ligase buffers Applications • Labeling 5 termini of nucleic acids to be used as Probes for hybridization Probes for transcript mapping Markers for gel electrophoresis Primers for DNA sequencing Primers for PCR • 5 phosphorylation of oligonucleotides PCR products other DNA or RNA prior to ligation • Phosphorylation of PCR primers • Detection of DNA modification by the 32P postlabeling assay • Removal of 3 phosphate groups Notes • The 5 termini of nucleic acids can be labeled by either the forward or the exchange reaction • Polyethylene glycol PEG and spermidine improve the rate and efficiency of the phosphorylation reaction PEG is used in the exchange reaction mixture • Since T4 Polynucleotide Kinase is inhibited by ammonium ions use sodium acetate to precipitate DNA prior to phosphorylation
    Catalog Number:
    ek0031
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher t4 pnk
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    Thermo Scientific T4 Polynucleotide Kinase T4 PNK catalyzes the transfer of the gamma phosphate from ATP to the 5 OH group of single and double stranded DNAs and RNAs oligonucleotides or nucleoside 3 monophosphates forward reaction The reaction is reversible In the presence of ADP T4 Polynucleotide Kinase exhibits 5 phosphatase activity and catalyzes the exchange of phosphate groups between 5 P oligo polynucleotides and ATP exchange reaction The enzyme is also a 3 phosphatase Highlights • Active in Thermo Scientific restriction enzyme RT and T4 DNA ligase buffers Applications • Labeling 5 termini of nucleic acids to be used as Probes for hybridization Probes for transcript mapping Markers for gel electrophoresis Primers for DNA sequencing Primers for PCR • 5 phosphorylation of oligonucleotides PCR products other DNA or RNA prior to ligation • Phosphorylation of PCR primers • Detection of DNA modification by the 32P postlabeling assay • Removal of 3 phosphate groups Notes • The 5 termini of nucleic acids can be labeled by either the forward or the exchange reaction • Polyethylene glycol PEG and spermidine improve the rate and efficiency of the phosphorylation reaction PEG is used in the exchange reaction mixture • Since T4 Polynucleotide Kinase is inhibited by ammonium ions use sodium acetate to precipitate DNA prior to phosphorylation
    https://www.bioz.com/result/t4 pnk/product/Thermo Fisher
    Average 99 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    t4 pnk - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells"

    Article Title: The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2018090914

    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    Figure Legend Snippet: RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Techniques Used: Selection, Software, RNA Binding Assay, Expressing, Imaging, Clone Assay, Immunoprecipitation, Labeling, Autoradiography, Western Blot

    2) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    3) Product Images from "DNA-guided DNA interference by a prokaryotic Argonaute"

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute

    Journal: Nature

    doi: 10.1038/nature12971

    Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a
    Figure Legend Snippet: Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a

    Techniques Used: Plasmid Preparation, Transformation Assay, Mutagenesis, Expressing, Purification

    4) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    Related Articles

    Ancient DNA Assay:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Ligation:

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    Article Snippet: .. The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Labeling:

    Article Title: Characterization of oligodeoxyribonucleotide synthesis on glass plates
    Article Snippet: .. A portion of the sample (3 µl) was labeled with [γ-32 P]ATP (5 µCi, 3000 Ci/mmol) using T4 polynucleotide kinase (1 U) and the conditions recommended by the manufacturer (Gibco). ..

    Purification:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE). .. Labelled nucleic acids were incubated with nucleases (DNase-free RNase A (Fermentas),RQ1 RNase-free DNaseI (Promega) or P1 nuclease (Sigma)) for 1 h at 37 °C.

    Sequencing:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Incubation:

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum
    Article Snippet: .. After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C. .. After protein extraction with phenol and chloroform, RNA was precipitated with ethanol.

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    Article Snippet: .. The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Mass Spectrometry:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: .. A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Polymerase Chain Reaction:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

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    Thermo Fisher t4 pnk
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
    T4 Pnk, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 973 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

    doi: 10.1681/ASN.2018090914

    Figure Lengend Snippet: RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Article Snippet: Beads were resuspended in PNK buffer containing 5 mM DTT, 0.2 μ Ci/ μ l ( γ 32 P)ATP (Hartmann-Analytic), and 1 U/ μ l T4 PNK (ThermoFisher).

    Techniques: Selection, Software, RNA Binding Assay, Expressing, Imaging, Clone Assay, Immunoprecipitation, Labeling, Autoradiography, Western Blot

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Techniques: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis

    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Journal: Nature

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    doi: 10.1038/s41586-018-0557-5

    Figure Lengend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Article Snippet: For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Techniques: Thin Layer Chromatography, Generated, Incubation