Structured Review

GE Healthcare α 32 p atp
A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled <t>ATP-FAM.</t> (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.
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1) Product Images from "Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities"

Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-01-0070

A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled ATP-FAM. (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.
Figure Legend Snippet: A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled ATP-FAM. (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.

Techniques Used: Mutagenesis, Activity Assay, Fluorescence, Binding Assay, Labeling, Centrifugation, SDS Page, Staining, Quantitation Assay

2) Product Images from "EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus "

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus

Journal: Journal of Bacteriology

doi: 10.1128/JB.00265-08

Analysis of EspA kinase activity. (A) Autoradiograph of GST-EspA kin (lane 1), GST-EspA kin ( H407A ) (lane 2), GST-EspA kin-rec (lane 3), and GST-EspA kin-rec ( D696A ) (lane 4) incubated in the presence of [γ- 32 P]ATP. (B) Coomassie-stained gel corresponding to panel A. (C) TLC analysis of [α- 32 P]ADP produced by [α- 32 P]ATP (lane 1), GST-EspA kin (lane 2), GST-EspA kin-rec (lane 3), and GST-EspA kin-rec ( D696A ) (lane 4). (D) Quantification of [α- 32 P]ADP signal in panel C as an average from triplicate experiments.
Figure Legend Snippet: Analysis of EspA kinase activity. (A) Autoradiograph of GST-EspA kin (lane 1), GST-EspA kin ( H407A ) (lane 2), GST-EspA kin-rec (lane 3), and GST-EspA kin-rec ( D696A ) (lane 4) incubated in the presence of [γ- 32 P]ATP. (B) Coomassie-stained gel corresponding to panel A. (C) TLC analysis of [α- 32 P]ADP produced by [α- 32 P]ATP (lane 1), GST-EspA kin (lane 2), GST-EspA kin-rec (lane 3), and GST-EspA kin-rec ( D696A ) (lane 4). (D) Quantification of [α- 32 P]ADP signal in panel C as an average from triplicate experiments.

Techniques Used: Activity Assay, Autoradiography, Incubation, Staining, Thin Layer Chromatography, Produced

3) Product Images from "Assays for transfer RNA-dependent amino acid biosynthesis"

Article Title: Assays for transfer RNA-dependent amino acid biosynthesis

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2007.06.010

[ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-
Figure Legend Snippet: [ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-

Techniques Used: Labeling

4) Product Images from "Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination"

Article Title: Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp096

Effects of wild-type and mutant UvsY proteins on ADP production by ( A ) UvsX-H195A mutant and ( B ) UvsX-H195Q mutant ssDNA-dependent ATPase activities. Velocities of ADP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM recombinase, 4.5 µM ssDNA, 4 mM α-[ 32 P]-ATP and variable concentrations of either UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds). All other conditions were as described in Materials and Methods section.
Figure Legend Snippet: Effects of wild-type and mutant UvsY proteins on ADP production by ( A ) UvsX-H195A mutant and ( B ) UvsX-H195Q mutant ssDNA-dependent ATPase activities. Velocities of ADP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM recombinase, 4.5 µM ssDNA, 4 mM α-[ 32 P]-ATP and variable concentrations of either UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds). All other conditions were as described in Materials and Methods section.

Techniques Used: Mutagenesis, Thin Layer Chromatography

Effects of wild-type and mutant UvsY proteins on ADP and AMP production by UvsX ssDNA-dependent ATPase activity. Velocities of ADP and AMP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM UvsX, 4.5 µM ssDNA and 4 mM α-[ 32 P]-ATP. All other conditions were as described in Materials and Methods section. ( A ) Velocity of ADP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. ( B ) Velocity of AMP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. Note that (B) is plotted on an expanded scale compared to (A). ( C ) ADP/AMP product ratio for wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration.
Figure Legend Snippet: Effects of wild-type and mutant UvsY proteins on ADP and AMP production by UvsX ssDNA-dependent ATPase activity. Velocities of ADP and AMP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM UvsX, 4.5 µM ssDNA and 4 mM α-[ 32 P]-ATP. All other conditions were as described in Materials and Methods section. ( A ) Velocity of ADP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. ( B ) Velocity of AMP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. Note that (B) is plotted on an expanded scale compared to (A). ( C ) ADP/AMP product ratio for wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration.

Techniques Used: Mutagenesis, Activity Assay, Thin Layer Chromatography, Concentration Assay

5) Product Images from "Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity"

Article Title: Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity

Journal: Cell Stress & Chaperones

doi:

Small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) inhibits the adenosine triphosphatase (ATPase) activity dependent on the constitutive counterpart, Hsc70, of heat shock protein (Hsp) 70. (A) Hsc70 (28 nM) was incubated with 0, 12, 58, 118, or 176 nM SGT/UBP or bovine serum albumin (BSA) and 1 μCi of [α- 32 P]ATP (13 nM) for 1 hour at 30°C. Adenosine diphosphate was separated from ATP by thin-layer chromatography and quantified using a Phosphorimager. Data are represented as the percent total ATPase activity, with the Hsc70 ATPase activity in the absence of BSA or SGT/UBP set to 100%. The Hsc70 ATPase activity in the presence of BSA (diamonds) or SGT/UBP (squares) is graphed as shown. (B) Hsc70 (28 nM) was incubated with 1 μCi of [α- 32 P]ATP with or without SGT/UBP at a final concentration of 28 nM. Hydrolysis of ATP was monitored in the presence of Hsc70 (triangle), Hsc70 and SGT/UBP (circle), SGT/UBP alone (squares), or with no protein (diamond). The data are represented as the percent ATP hydrolysis and are plotted as a function of time from 0 minutes to 75 minutes
Figure Legend Snippet: Small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) inhibits the adenosine triphosphatase (ATPase) activity dependent on the constitutive counterpart, Hsc70, of heat shock protein (Hsp) 70. (A) Hsc70 (28 nM) was incubated with 0, 12, 58, 118, or 176 nM SGT/UBP or bovine serum albumin (BSA) and 1 μCi of [α- 32 P]ATP (13 nM) for 1 hour at 30°C. Adenosine diphosphate was separated from ATP by thin-layer chromatography and quantified using a Phosphorimager. Data are represented as the percent total ATPase activity, with the Hsc70 ATPase activity in the absence of BSA or SGT/UBP set to 100%. The Hsc70 ATPase activity in the presence of BSA (diamonds) or SGT/UBP (squares) is graphed as shown. (B) Hsc70 (28 nM) was incubated with 1 μCi of [α- 32 P]ATP with or without SGT/UBP at a final concentration of 28 nM. Hydrolysis of ATP was monitored in the presence of Hsc70 (triangle), Hsc70 and SGT/UBP (circle), SGT/UBP alone (squares), or with no protein (diamond). The data are represented as the percent ATP hydrolysis and are plotted as a function of time from 0 minutes to 75 minutes

Techniques Used: Binding Assay, Activity Assay, Incubation, Thin Layer Chromatography, Concentration Assay

6) Product Images from "Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination"

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.41.9.4217-4223.2003

(A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α- 32 P]ATP was hydrolyzed to [α- 32 P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α- 32 P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32 P-labeled template RNA was loaded as a size control (lane 2).
Figure Legend Snippet: (A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α- 32 P]ATP was hydrolyzed to [α- 32 P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α- 32 P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32 P-labeled template RNA was loaded as a size control (lane 2).

Techniques Used: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, Staining, Activity Assay, Labeling, Autoradiography

7) Product Images from "Foot-and-Mouth Disease Virus Mutant with Decreased Sensitivity to Ribavirin: Implications for Error Catastrophe ▿"

Article Title: Foot-and-Mouth Disease Virus Mutant with Decreased Sensitivity to Ribavirin: Implications for Error Catastrophe ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01606-06

Incorporation of RMP relative to that of GMP or AMP by purified FMDV 3D(M) or FMDV 3D(M-M296I) using homopolymeric templates-primers. (A) Incorporation of RMP and GMP using poly(rC)-oligo(dG) 15 as the template-primer. The reaction was performed in Mg(CH 3 COO) 2 (15 mM), [α- 32 P]GTP (1 nM), and increasing concentrations of RTP (50, 100, 250, and 500 μM). Results of thin-layer chromatography of RNase-digested products are shown; the black vertical arrow shows the direction of the migration, and the horizontal arrow indicates the sample application point. The positions of ribavirin monophosphate (R) and GMP (G) are indicated. Ribavirin monophosphate control migration is the product of RMP incorporation at position +1 (from the primer 3′ end) into sym/sub-RNA 5′-GUACGGGCCC-3′, with [α- 32 P]UTP incorporated at position +2. RNase digestion and nucleotide (nearest-neighbor) analysis were performed as detailed in Materials and Methods. (B) Incorporation of RMP as a function of RTP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results from four experiments performed like that whose results are shown in panel A. (C) Incorporation of RMP and AMP using poly(rU)-poly(rA) 6 as the template-primer, MnCl 2 (10 mM), [α- 32 P] ATP (1 nM), and increasing concentrations of RTP (62.5, 125, 250, and 500 μM). The results of thin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and AMP (A) are indicated; other symbols are as described for panel A. (D) Incorporation of RMP as a function of RTP concentration [○, 3D(M); •, 3D(M-M296I)]. Averages of results from three experiments performed like that whose results are shown in panel C (note the different scales in the ordinates between panels B and D). (E) Incorporation of RMP and GMP using poly(rC)-oligo(dG) 15 as the template-primer, MnCl 2 (10 mM), RTP (400 μM), and increasing concentrations of [α- 32 P]GTP (0.5, 5, 10, and 50 μM). The results of hin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and GMP (G) are indicated. The N lane shows the position of nucleoside monophosphates. Other symbols are as described for panel A. (F) Incorporation of RMP as a function of GTP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results of three experiments performed like that whose results are shown in panel E. (G) Incorporation of RMP and AMP using poly(rU)-poly(rA) 6 as the template-primer, MnCl 2 (10 mM), RTP (400 μM), and increasing concentrations of [α- 32 P]ATP (0.05, 0.5, 1, and 5 μM). Results of thin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and AMP (A) are indicated. Other symbols are as described for panel A. (H) Incorporation of RMP as a function of ATP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results from three experiments performed like that whose results are shown in panel G (note the different scales in the ordinates between panels F and H). Standard deviations of densitometry values are given. Procedures are detailed in Materials and Methods.
Figure Legend Snippet: Incorporation of RMP relative to that of GMP or AMP by purified FMDV 3D(M) or FMDV 3D(M-M296I) using homopolymeric templates-primers. (A) Incorporation of RMP and GMP using poly(rC)-oligo(dG) 15 as the template-primer. The reaction was performed in Mg(CH 3 COO) 2 (15 mM), [α- 32 P]GTP (1 nM), and increasing concentrations of RTP (50, 100, 250, and 500 μM). Results of thin-layer chromatography of RNase-digested products are shown; the black vertical arrow shows the direction of the migration, and the horizontal arrow indicates the sample application point. The positions of ribavirin monophosphate (R) and GMP (G) are indicated. Ribavirin monophosphate control migration is the product of RMP incorporation at position +1 (from the primer 3′ end) into sym/sub-RNA 5′-GUACGGGCCC-3′, with [α- 32 P]UTP incorporated at position +2. RNase digestion and nucleotide (nearest-neighbor) analysis were performed as detailed in Materials and Methods. (B) Incorporation of RMP as a function of RTP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results from four experiments performed like that whose results are shown in panel A. (C) Incorporation of RMP and AMP using poly(rU)-poly(rA) 6 as the template-primer, MnCl 2 (10 mM), [α- 32 P] ATP (1 nM), and increasing concentrations of RTP (62.5, 125, 250, and 500 μM). The results of thin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and AMP (A) are indicated; other symbols are as described for panel A. (D) Incorporation of RMP as a function of RTP concentration [○, 3D(M); •, 3D(M-M296I)]. Averages of results from three experiments performed like that whose results are shown in panel C (note the different scales in the ordinates between panels B and D). (E) Incorporation of RMP and GMP using poly(rC)-oligo(dG) 15 as the template-primer, MnCl 2 (10 mM), RTP (400 μM), and increasing concentrations of [α- 32 P]GTP (0.5, 5, 10, and 50 μM). The results of hin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and GMP (G) are indicated. The N lane shows the position of nucleoside monophosphates. Other symbols are as described for panel A. (F) Incorporation of RMP as a function of GTP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results of three experiments performed like that whose results are shown in panel E. (G) Incorporation of RMP and AMP using poly(rU)-poly(rA) 6 as the template-primer, MnCl 2 (10 mM), RTP (400 μM), and increasing concentrations of [α- 32 P]ATP (0.05, 0.5, 1, and 5 μM). Results of thin-layer chromatography of RNase-digested products are shown. The positions of ribavirin monophosphate (R) and AMP (A) are indicated. Other symbols are as described for panel A. (H) Incorporation of RMP as a function of ATP concentration [○, 3D(M); •, 3D(M-M296I)]. Shown are averages of results from three experiments performed like that whose results are shown in panel G (note the different scales in the ordinates between panels F and H). Standard deviations of densitometry values are given. Procedures are detailed in Materials and Methods.

Techniques Used: Purification, Thin Layer Chromatography, Migration, Concentration Assay, Chromatography

8) Product Images from "Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I"

Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.
Figure Legend Snippet: T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

Techniques Used: Activity Assay, Incubation, Inhibition, Electrophoresis, Autoradiography

9) Product Images from "Primase Directs the Release of DnaC from DnaB"

Article Title: Primase Directs the Release of DnaC from DnaB

Journal: Molecular cell

doi: 10.1016/j.molcel.2009.12.031

R216A and R220A are active in ATP binding, which stimulates their ability to interact with DnaB ( A ) UV crosslinking of [α- 32 ), but only one of each pair is shown because the results are essentially identical. As negative controls, BSA (200 ng) was used in place of wild type DnaC, or wild type DnaC (200 ng) and [α- 32 P] ATP were incubated with 0.1 mM ATP. The electrophoretic mobilities of BSA and DnaC, which were visualized by staining with Coomassie Brilliant Blue before autoradiography, are indicated at the right. ( B , C ). After protein immobilization using a Bio-Rad dot blot apparatus, the filters (BA-85, Whatman) were incubated with DnaB (1 μg/ml) in buffer (40 mM Hepes-KOH pH 8.0, 20 mM Tris-HCl, pH 7.6, 10 mM magnesium chloride, 4% sucrose, 4 mM DTT, and 0.1 mg/ml BSA) lacking or supplemented with 1 mM ATP. The washed filters were then incubated with affinity-purified antibody that specifically recognizes DnaB. Immune complexes were detected as described in Experimental Procedures .
Figure Legend Snippet: R216A and R220A are active in ATP binding, which stimulates their ability to interact with DnaB ( A ) UV crosslinking of [α- 32 ), but only one of each pair is shown because the results are essentially identical. As negative controls, BSA (200 ng) was used in place of wild type DnaC, or wild type DnaC (200 ng) and [α- 32 P] ATP were incubated with 0.1 mM ATP. The electrophoretic mobilities of BSA and DnaC, which were visualized by staining with Coomassie Brilliant Blue before autoradiography, are indicated at the right. ( B , C ). After protein immobilization using a Bio-Rad dot blot apparatus, the filters (BA-85, Whatman) were incubated with DnaB (1 μg/ml) in buffer (40 mM Hepes-KOH pH 8.0, 20 mM Tris-HCl, pH 7.6, 10 mM magnesium chloride, 4% sucrose, 4 mM DTT, and 0.1 mg/ml BSA) lacking or supplemented with 1 mM ATP. The washed filters were then incubated with affinity-purified antibody that specifically recognizes DnaB. Immune complexes were detected as described in Experimental Procedures .

Techniques Used: Binding Assay, Incubation, Staining, Autoradiography, Dot Blot, Affinity Purification

10) Product Images from "Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme"

Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

Journal:

doi: 10.1038/emboj.2008.124

CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows
Figure Legend Snippet: CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

Techniques Used: Mutagenesis

11) Product Images from "Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase"

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1133325100

( A and B ) Effect of template length on Eco SBB activation. Runoff transcription reactions contained 1 nM template, 1 μM Eco SSB, and 1 μM mini-vRNAP and were incubated for 10 min at 37°C. ( C ) Interactions of the nascent transcript with mini-vRNAP and Eco SSB. ( Upper ) DNA template and nascent RNA arrangement in SEC. DNA templates bearing a single A in the transcribed region at the indicated positions numbered relative to the transcription start site (+1) were used in mini-vRNAP transcription reactions with 5-I-UTP, GTP, and [α- 32 P]ATP. Arrowhead, position of the active center in the SEC. RNA is represented by a line. IU at positions –10 and –17 of the RNA in SEC is shown. ( Lower ) Crosslinking of the nascent RNA containing IU at different positions to mini-vRNAP and Eco SSB in SEC. Positions containing IU in nascent RNA of the SEC are numbered relative to the 3′ end (–1). Arrows indicate RNA crosslinked to mini-vRNAP and to Eco SSB. IU, 5-iodo-uridine.
Figure Legend Snippet: ( A and B ) Effect of template length on Eco SBB activation. Runoff transcription reactions contained 1 nM template, 1 μM Eco SSB, and 1 μM mini-vRNAP and were incubated for 10 min at 37°C. ( C ) Interactions of the nascent transcript with mini-vRNAP and Eco SSB. ( Upper ) DNA template and nascent RNA arrangement in SEC. DNA templates bearing a single A in the transcribed region at the indicated positions numbered relative to the transcription start site (+1) were used in mini-vRNAP transcription reactions with 5-I-UTP, GTP, and [α- 32 P]ATP. Arrowhead, position of the active center in the SEC. RNA is represented by a line. IU at positions –10 and –17 of the RNA in SEC is shown. ( Lower ) Crosslinking of the nascent RNA containing IU at different positions to mini-vRNAP and Eco SSB in SEC. Positions containing IU in nascent RNA of the SEC are numbered relative to the 3′ end (–1). Arrows indicate RNA crosslinked to mini-vRNAP and to Eco SSB. IU, 5-iodo-uridine.

Techniques Used: Activation Assay, Incubation, Size-exclusion Chromatography

12) Product Images from "The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP"

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP

Journal: RNA

doi: 10.1261/rna.2110805

Position 56 is specifically methylated by the P. abyssi enzyme. T7 in vitro transcripts of E. coli wild type tRNA Leu2 and P. abyssi tRNA Leu/CAA , uniformly labeled by incorporation of [α- 32 P]-NTP were incubated for 1 h at 50°C in the presence of the purified P. abyssi protein. ( A ) 2D TLC in system A+C of RNase T2 digests obtained from E. coli tRNA Leu2 labeled with ATP, CTP, GTP, or UTP as indicated. ( B ) 2D TLC of RNase T2 digest of P. abyssi tRNA LeuCAA labeled with ATP. The yield of methylation is indicated on each chromatogram. A cloverleaf representation of the corresponding tRNAs analyzed is shown on the right with CA dinucleosides marked in gray. ( C ) Comparative kinetic analysis of a Trm56-catalyzed methylation of two tRNA substrates, E. coli tRNA Leu2 , and P. abyssi tRNA Leu/CAA . tRNAs uniformly labeled by incorporation of [α- 32 P]-ATP were incubated for different period of times as described above. The yield of methylation was determined by quantitative analysis of 2D-TLC.
Figure Legend Snippet: Position 56 is specifically methylated by the P. abyssi enzyme. T7 in vitro transcripts of E. coli wild type tRNA Leu2 and P. abyssi tRNA Leu/CAA , uniformly labeled by incorporation of [α- 32 P]-NTP were incubated for 1 h at 50°C in the presence of the purified P. abyssi protein. ( A ) 2D TLC in system A+C of RNase T2 digests obtained from E. coli tRNA Leu2 labeled with ATP, CTP, GTP, or UTP as indicated. ( B ) 2D TLC of RNase T2 digest of P. abyssi tRNA LeuCAA labeled with ATP. The yield of methylation is indicated on each chromatogram. A cloverleaf representation of the corresponding tRNAs analyzed is shown on the right with CA dinucleosides marked in gray. ( C ) Comparative kinetic analysis of a Trm56-catalyzed methylation of two tRNA substrates, E. coli tRNA Leu2 , and P. abyssi tRNA Leu/CAA . tRNAs uniformly labeled by incorporation of [α- 32 P]-ATP were incubated for different period of times as described above. The yield of methylation was determined by quantitative analysis of 2D-TLC.

Techniques Used: Methylation, In Vitro, Cellular Antioxidant Activity Assay, Labeling, Incubation, Purification, Thin Layer Chromatography

13) Product Images from "Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41"

Article Title: Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41

Journal: PLoS ONE

doi: 10.1371/journal.pone.0136879

Comparisons of promoter strength and structure. (A). Sequences and structures of the 65-bp constructs contain a linear promoter and a pre-melted region with or without 14S rRNA promoter. (B-D). transcripts yielded from the ab0ove constructs using Rpo41 wild-type or ΔN160 (0.5 μM) in the absence or presence of Mtf1 (2.5 μM), labeled with [γ- 32 P]GTP, [γ- 32 P]ATP, and [α- 32 P]ATP, respectively.
Figure Legend Snippet: Comparisons of promoter strength and structure. (A). Sequences and structures of the 65-bp constructs contain a linear promoter and a pre-melted region with or without 14S rRNA promoter. (B-D). transcripts yielded from the ab0ove constructs using Rpo41 wild-type or ΔN160 (0.5 μM) in the absence or presence of Mtf1 (2.5 μM), labeled with [γ- 32 P]GTP, [γ- 32 P]ATP, and [α- 32 P]ATP, respectively.

Techniques Used: Construct, Labeling

Activities of Rpo41 variants on pre-melted promoter-containing DNA. (A). The duplex DNA template contains a 14S rRNA promoter, with non-complementary sequences from positions-4 to +2. (B) (1) [γ- 32 P]ATP labeled transcripts on the above template (1 μM) using Rpo41 (0.5 μM wild-type or a variant) in the absence or presence of Mtf1 (2.5 μM) were visualized on a polyacrylamide denaturing gel. (2) Same as (1), except that transcripts are labeled with [γ- 32 P]ATP, [γ- 32 P]GTP or [α- 32 P]ATP. C) The intensities of run-off transcripts in B (1) quantified using Quantity One software.
Figure Legend Snippet: Activities of Rpo41 variants on pre-melted promoter-containing DNA. (A). The duplex DNA template contains a 14S rRNA promoter, with non-complementary sequences from positions-4 to +2. (B) (1) [γ- 32 P]ATP labeled transcripts on the above template (1 μM) using Rpo41 (0.5 μM wild-type or a variant) in the absence or presence of Mtf1 (2.5 μM) were visualized on a polyacrylamide denaturing gel. (2) Same as (1), except that transcripts are labeled with [γ- 32 P]ATP, [γ- 32 P]GTP or [α- 32 P]ATP. C) The intensities of run-off transcripts in B (1) quantified using Quantity One software.

Techniques Used: Labeling, Variant Assay, Software

14) Product Images from "Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC"

Article Title: Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1319353111

KaiA-stimulated nucleotide exchange on KaiC. ( A and B ) KaiC-bound radioactive nucleotides were chased with exogenous nonradioactive ATP. KaiC hexamers were prepared from monomers in the presence of 2 mM [α- 32 P]ATP, unbound nucleotides were removed,
Figure Legend Snippet: KaiA-stimulated nucleotide exchange on KaiC. ( A and B ) KaiC-bound radioactive nucleotides were chased with exogenous nonradioactive ATP. KaiC hexamers were prepared from monomers in the presence of 2 mM [α- 32 P]ATP, unbound nucleotides were removed,

Techniques Used:

Effect of KaiA on the nucleotide-bound state of KaiC. ( A ) Full-length KaiC hexamers were reconstituted from monomers in the presence of 2 mM [α- 32 P]ATP and incubated at 30 °C in the presence (○) or absence (●) of KaiA.
Figure Legend Snippet: Effect of KaiA on the nucleotide-bound state of KaiC. ( A ) Full-length KaiC hexamers were reconstituted from monomers in the presence of 2 mM [α- 32 P]ATP and incubated at 30 °C in the presence (○) or absence (●) of KaiA.

Techniques Used: Incubation

15) Product Images from "Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase"

Article Title: Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Journal: Journal of Virology

doi: 10.1128/JVI.75.17.8289-8297.2001

ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [α- 32 P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS–12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.
Figure Legend Snippet: ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [α- 32 P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS–12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Autoradiography

16) Product Images from "Assays for transfer RNA-dependent amino acid biosynthesis"

Article Title: Assays for transfer RNA-dependent amino acid biosynthesis

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2007.06.010

[ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-
Figure Legend Snippet: [ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-

Techniques Used: Labeling

17) Product Images from "Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase"

Article Title: Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn036

tRNA Ser chimera with transplanted (tRNA Sec ) identity elements induces the ATPase activity of PSTK. A graph is shown of the ratio of [α- 32 P]ATP converted to [α- 32 P]ADP by PSTK (1 µM) in the presence of 1 µM tRNA Sec , chimera tRNA, G2-C71:C-G tRNA Sec mutant or tRNA Ser . The minimal ATPase activity in the absence of tRNA was subtracted. Error bars represent the standard deviation of three separate experiments.
Figure Legend Snippet: tRNA Ser chimera with transplanted (tRNA Sec ) identity elements induces the ATPase activity of PSTK. A graph is shown of the ratio of [α- 32 P]ATP converted to [α- 32 P]ADP by PSTK (1 µM) in the presence of 1 µM tRNA Sec , chimera tRNA, G2-C71:C-G tRNA Sec mutant or tRNA Ser . The minimal ATPase activity in the absence of tRNA was subtracted. Error bars represent the standard deviation of three separate experiments.

Techniques Used: Size-exclusion Chromatography, Activity Assay, Mutagenesis, Standard Deviation

18) Product Images from "Rice Small C2-Domain Proteins Are Phosphorylated by Calcium-Dependent Protein Kinase"

Article Title: Rice Small C2-Domain Proteins Are Phosphorylated by Calcium-Dependent Protein Kinase

Journal: Molecules and Cells

doi: 10.1007/s10059-013-2185-0

In vitro phosphorylation on serine 41 of OsERG1 by OsCDPK5. (A) Prediction of a putative phosphorylation site on C2 domains by serine/threonine protein kinases. Serine (S) or threonine (T) residues that are conserved in C2-domain proteins, which could serve as phosphorylation sites for serine/threonine protein kinases, are indicated with the asterisk. Amino acid residues identical to the consensus sequence are shown in dark. (B) Mutational analysis for phosphorylation of GST-OsERG1 by OsCDPK5. Wild-type and mutant proteins of GST-OsERG1 (WT), GST-OsERG1 (S41A), GST-OsERG1(S42A), and GST-OsERG1(S41D) constructs and GST-OsCDPK5 recombinant proteins (1 μg) were incubated for 30 min at 30°C in 25 μl kinase buffer containing 10 μCi of [α- 32 P]ATP in the presence of 1 mM calcium ions. Identical samples were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (right panel).
Figure Legend Snippet: In vitro phosphorylation on serine 41 of OsERG1 by OsCDPK5. (A) Prediction of a putative phosphorylation site on C2 domains by serine/threonine protein kinases. Serine (S) or threonine (T) residues that are conserved in C2-domain proteins, which could serve as phosphorylation sites for serine/threonine protein kinases, are indicated with the asterisk. Amino acid residues identical to the consensus sequence are shown in dark. (B) Mutational analysis for phosphorylation of GST-OsERG1 by OsCDPK5. Wild-type and mutant proteins of GST-OsERG1 (WT), GST-OsERG1 (S41A), GST-OsERG1(S42A), and GST-OsERG1(S41D) constructs and GST-OsCDPK5 recombinant proteins (1 μg) were incubated for 30 min at 30°C in 25 μl kinase buffer containing 10 μCi of [α- 32 P]ATP in the presence of 1 mM calcium ions. Identical samples were separated by 13% SDS-PAGE and stained with Coomassie brilliant blue (right panel).

Techniques Used: In Vitro, Sequencing, Mutagenesis, Construct, Recombinant, Incubation, SDS Page, Staining

19) Product Images from "Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes"

Article Title: Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0050004

In Vitro ATP Hydrolysis Assay of Selenophosphate Synthetase and NMR Spectroscopic Analysis Cloning of the genes, mouse sps1, mouse sps2, SelD, and C. elegans sps2, and mutation of mouse sps2 to sps2-Cys and reaction conditions are detailed in Materials and Methods. For NMR analysis, 200 μl of ATP hydrolysis reaction was sealed in 3-mm NMR tubes and incubated at 37 °C for 4 h before 31 P NMR spectroscopic analysis [ 9 ]. (A) Complete 31 P-NMR spectra of ATP hydrolysis products generated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, and mSPS1 are shown. (B) Expanded spectra of the ordinate and abscissa between 15 and 30 ppm for these products are shown. (C) ATP hydrolysis reactions with [α- 32 P]ATP, either without or with 0.25 mM selenide, incubated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, or mSPS1; at the end of the incubation period, reactions were loaded onto PEI TLC plates, run in 0.8 M LiCl, and exposed to a PhosphorImager screen as described in Materials and Methods.
Figure Legend Snippet: In Vitro ATP Hydrolysis Assay of Selenophosphate Synthetase and NMR Spectroscopic Analysis Cloning of the genes, mouse sps1, mouse sps2, SelD, and C. elegans sps2, and mutation of mouse sps2 to sps2-Cys and reaction conditions are detailed in Materials and Methods. For NMR analysis, 200 μl of ATP hydrolysis reaction was sealed in 3-mm NMR tubes and incubated at 37 °C for 4 h before 31 P NMR spectroscopic analysis [ 9 ]. (A) Complete 31 P-NMR spectra of ATP hydrolysis products generated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, and mSPS1 are shown. (B) Expanded spectra of the ordinate and abscissa between 15 and 30 ppm for these products are shown. (C) ATP hydrolysis reactions with [α- 32 P]ATP, either without or with 0.25 mM selenide, incubated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, or mSPS1; at the end of the incubation period, reactions were loaded onto PEI TLC plates, run in 0.8 M LiCl, and exposed to a PhosphorImager screen as described in Materials and Methods.

Techniques Used: In Vitro, Hydrolysis Assay, Nuclear Magnetic Resonance, Clone Assay, Mutagenesis, Incubation, Generated, Thin Layer Chromatography

20) Product Images from "Murine Pancreatic Beta TC3 Cells Show Greater 2?, 5?-Oligoadenylate Synthetase (2?5?AS) Antiviral Enzyme Activity and Apoptosis Following IFN-α or Poly(I:C) Treatment than Pancreatic Alpha TC3 Cells"

Article Title: Murine Pancreatic Beta TC3 Cells Show Greater 2?, 5?-Oligoadenylate Synthetase (2?5?AS) Antiviral Enzyme Activity and Apoptosis Following IFN-α or Poly(I:C) Treatment than Pancreatic Alpha TC3 Cells

Journal: Experimental Diabetes Research

doi: 10.1155/2009/631026

Enzyme activity of 2′5′AS in non-stimulated and IFN- α stimulated beta TC3 and alpha TC3 cells. Beta TC3 (a) and alpha TC3 (b) cells were incubated in the presence or absence of 500 U/ml of IFN- α . After the indicated time periods, cell lysates were prepared. The lysates were incubated with 0.15 μ Ci α 32 P-ATP for 120 minutes at 30°C in the presence of 100 μ g/mL poly(I:C), and then separated by ascending TLC. The generated 2′5′A were quantitated by PhosphorImager analysis. Enzyme activity was expressed in units per milligram ( n = 4, standard deviations varied between 6–13%).
Figure Legend Snippet: Enzyme activity of 2′5′AS in non-stimulated and IFN- α stimulated beta TC3 and alpha TC3 cells. Beta TC3 (a) and alpha TC3 (b) cells were incubated in the presence or absence of 500 U/ml of IFN- α . After the indicated time periods, cell lysates were prepared. The lysates were incubated with 0.15 μ Ci α 32 P-ATP for 120 minutes at 30°C in the presence of 100 μ g/mL poly(I:C), and then separated by ascending TLC. The generated 2′5′A were quantitated by PhosphorImager analysis. Enzyme activity was expressed in units per milligram ( n = 4, standard deviations varied between 6–13%).

Techniques Used: Activity Assay, Incubation, Thin Layer Chromatography, Generated

Protein expression and activity of 2′5′AS in non-stimulated and IFN- α stimulated beta TC3 and alpha TC3 cells. After 24 hours of IFN- α stimulation, cell lysates were prepared. (a) For immunoblot, cell extracts were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The polyclonal antibody specific to human 2′5′AS (cross-reactive with murine 2′5′AS) was used to detect the expressed proteins. The polyclonal antibody against mouse GAPDH was used to quantitate the loading amounts; (b) For assay of 2′5′AS activity, lysates were incubated with 0.15 μ Ci α 32 P-ATP for 120 minutes at 30°C in the presence of 100 μ g/ml poly(I:C), and then separated by ascending TLC. The generated 2′5′-oligoadenylates (2′5′A) were quantitated by PhosphorImager analysis. Enzyme activity was expressed in units per milligram protein ( n = 3).
Figure Legend Snippet: Protein expression and activity of 2′5′AS in non-stimulated and IFN- α stimulated beta TC3 and alpha TC3 cells. After 24 hours of IFN- α stimulation, cell lysates were prepared. (a) For immunoblot, cell extracts were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. The polyclonal antibody specific to human 2′5′AS (cross-reactive with murine 2′5′AS) was used to detect the expressed proteins. The polyclonal antibody against mouse GAPDH was used to quantitate the loading amounts; (b) For assay of 2′5′AS activity, lysates were incubated with 0.15 μ Ci α 32 P-ATP for 120 minutes at 30°C in the presence of 100 μ g/ml poly(I:C), and then separated by ascending TLC. The generated 2′5′-oligoadenylates (2′5′A) were quantitated by PhosphorImager analysis. Enzyme activity was expressed in units per milligram protein ( n = 3).

Techniques Used: Expressing, Activity Assay, SDS Page, Incubation, Thin Layer Chromatography, Generated

21) Product Images from "A novel embryonic poly(A) binding protein, ePAB, regulates mRNA deadenylation in Xenopus egg extracts"

Article Title: A novel embryonic poly(A) binding protein, ePAB, regulates mRNA deadenylation in Xenopus egg extracts

Journal: Genes & Development

doi: 10.1101/gad.872201

ePAB expression during Xenopus early development. ( A ) Extract from various stages of development [1/2-egg equivalent per lane: stage VI oocyte, mature egg, 4-cell embryo (1.5 h), 16-cell embryo (2.5 h), morula (3.5 h), blastula (5 h), mid-blastula (7 h), gastrula (9 h), early neurula (12 h), 1-day embryo, and 30-h embryo] and XTC cells were fractionated by 15% PAGE and immunostained with anti-ePAB (third panel), monoclonal anti-PABP1 (top panel), or monoclonal anti-HuR, identifying ElrA (second panel), antibodies. The blot that was initially probed with anti-PABP1 was later reprobed for ePAB; the asterisk (*) marks the remaining signal from anti-PABP1, therefore indicating the mobility of PABP1 relative to ePAB. The same samples were also UV-crosslinked to [α- 32 P]ATP labeled AT-UTR p(A) + (fourth panel) and GC-UTR p(A) + (bottom panel). ( B ) Egg extract was crosslinked to: [α- 32 P]ATP-labeled AT-UTR p(A) + (lane 1 ), [α- 32 P]ATP-labeled GC-UTR p(A) + (lane 4 ), or [α- 32 P]UTP-labeled AT-UTR (lane 7 ). Crosslinked samples (Total) were immunoprecipitated with anti-ePAB (α-ePAB, lanes 3,6,9 ) or preimmune (α-Pre, lanes 2,5 , or 8 ) serum. The arrow denotes the ∼65-kD crosslinked band precipitated by anti-ePAB. ( C ) Stage VI oocytes were manually enucleated and 1/2-oocyte equivalents of the total (Tot), cytoplasmic (cyt), and nuclear (GV) fractions were immunostained with anti-ePAB, monoclonal anti-PABP1, and monoclonal anti-HuR, identifying ElrA, antibodies.
Figure Legend Snippet: ePAB expression during Xenopus early development. ( A ) Extract from various stages of development [1/2-egg equivalent per lane: stage VI oocyte, mature egg, 4-cell embryo (1.5 h), 16-cell embryo (2.5 h), morula (3.5 h), blastula (5 h), mid-blastula (7 h), gastrula (9 h), early neurula (12 h), 1-day embryo, and 30-h embryo] and XTC cells were fractionated by 15% PAGE and immunostained with anti-ePAB (third panel), monoclonal anti-PABP1 (top panel), or monoclonal anti-HuR, identifying ElrA (second panel), antibodies. The blot that was initially probed with anti-PABP1 was later reprobed for ePAB; the asterisk (*) marks the remaining signal from anti-PABP1, therefore indicating the mobility of PABP1 relative to ePAB. The same samples were also UV-crosslinked to [α- 32 P]ATP labeled AT-UTR p(A) + (fourth panel) and GC-UTR p(A) + (bottom panel). ( B ) Egg extract was crosslinked to: [α- 32 P]ATP-labeled AT-UTR p(A) + (lane 1 ), [α- 32 P]ATP-labeled GC-UTR p(A) + (lane 4 ), or [α- 32 P]UTP-labeled AT-UTR (lane 7 ). Crosslinked samples (Total) were immunoprecipitated with anti-ePAB (α-ePAB, lanes 3,6,9 ) or preimmune (α-Pre, lanes 2,5 , or 8 ) serum. The arrow denotes the ∼65-kD crosslinked band precipitated by anti-ePAB. ( C ) Stage VI oocytes were manually enucleated and 1/2-oocyte equivalents of the total (Tot), cytoplasmic (cyt), and nuclear (GV) fractions were immunostained with anti-ePAB, monoclonal anti-PABP1, and monoclonal anti-HuR, identifying ElrA, antibodies.

Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, Labeling, Immunoprecipitation

22) Product Images from "A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity"

Article Title: A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity

Journal: Journal of Bacteriology

doi:

Adenylation of Lig Tk with ATP or NAD + as a cofactor. Lig Tk was incubated at 80°C for 2 h with [α- 32 P]ATP or [adenylate- 32 P]NAD + , both with 32 P in the phosphate group of the AMP moieties. After incubation, the proteins were applied to SDS-PAGE gels and detected by autoradiography.
Figure Legend Snippet: Adenylation of Lig Tk with ATP or NAD + as a cofactor. Lig Tk was incubated at 80°C for 2 h with [α- 32 P]ATP or [adenylate- 32 P]NAD + , both with 32 P in the phosphate group of the AMP moieties. After incubation, the proteins were applied to SDS-PAGE gels and detected by autoradiography.

Techniques Used: Incubation, SDS Page, Autoradiography

23) Product Images from "Identification and Characterization of an RNA-Dependent RNA Polymerase Activity within the Nonstructural Protein 5B Region of Bovine Viral Diarrhea Virus"

Article Title: Identification and Characterization of an RNA-Dependent RNA Polymerase Activity within the Nonstructural Protein 5B Region of Bovine Viral Diarrhea Virus

Journal: Journal of Virology

doi:

(A) Effects of RNase A and actinomycin D on NS5B C-His RdRp products. Addition of either actinomycin D (Act D), nonradioactive NTP mixture (500 μM [each] CTP, GTP, and UTP and 20 μM ATP), or postreaction digestion by RNase A is indicated above the lanes. Products were extracted and separated on a formaldehyde-agarose gel as described in the text. Products representing a putative monomer and a dimer hairpin are indicated schematically. The positions of RNA molecular size markers are shown on the right. (B) TNTase activity of NS5B C-His . Polymerase reactions were performed using [α- 32 P]UTP, [α- 32 P]GTP, or [α- 32 P]ATP as indicated above the lanes. The absence (−) or presence (+) of the three remaining rNTPs in the reaction is also indicated above the lanes. Other details are as described for panel A. (C) Northern blot analysis of RdRp products. Polymerase reactions were performed using pTemp1 RNA in the presence (+) or absence (−) of all four unlabeled rNTPs. Reaction products were analyzed by Northern blot analysis using a [ 32 P]-labeled riboprobe specific for the negative strand. Other details are as described for panel A. (D) NS5B C-His activity on nonviral templates. Homopolymeric templates [poly(rC) or poly(dC)] or annealed primer-template pairs [poly(rC)-oligo(rG), poly(rC)-oligo(dG), poly(dC)-oligo(rG), or poly(dC)-oligo(dG)] were used in standard RdRp reactions. Reaction products were quantified by scintillation counting of acid-insoluble radioactivity. Activity without a template (−) was used to determine background radioactivity.
Figure Legend Snippet: (A) Effects of RNase A and actinomycin D on NS5B C-His RdRp products. Addition of either actinomycin D (Act D), nonradioactive NTP mixture (500 μM [each] CTP, GTP, and UTP and 20 μM ATP), or postreaction digestion by RNase A is indicated above the lanes. Products were extracted and separated on a formaldehyde-agarose gel as described in the text. Products representing a putative monomer and a dimer hairpin are indicated schematically. The positions of RNA molecular size markers are shown on the right. (B) TNTase activity of NS5B C-His . Polymerase reactions were performed using [α- 32 P]UTP, [α- 32 P]GTP, or [α- 32 P]ATP as indicated above the lanes. The absence (−) or presence (+) of the three remaining rNTPs in the reaction is also indicated above the lanes. Other details are as described for panel A. (C) Northern blot analysis of RdRp products. Polymerase reactions were performed using pTemp1 RNA in the presence (+) or absence (−) of all four unlabeled rNTPs. Reaction products were analyzed by Northern blot analysis using a [ 32 P]-labeled riboprobe specific for the negative strand. Other details are as described for panel A. (D) NS5B C-His activity on nonviral templates. Homopolymeric templates [poly(rC) or poly(dC)] or annealed primer-template pairs [poly(rC)-oligo(rG), poly(rC)-oligo(dG), poly(dC)-oligo(rG), or poly(dC)-oligo(dG)] were used in standard RdRp reactions. Reaction products were quantified by scintillation counting of acid-insoluble radioactivity. Activity without a template (−) was used to determine background radioactivity.

Techniques Used: Activated Clotting Time Assay, Agarose Gel Electrophoresis, Activity Assay, Northern Blot, Labeling, Radioactivity

24) Product Images from "Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation"

Article Title: Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm1134

ATPase activity of M. jannaschii PSTK. Graph of the ratio of [α- 32 P]ATP converted to [α- 32 P]ADP by PSTK (200 nM) in the absence or presence of 1 µM tRNA Sec , Ser-tRNA Sec , or Ser-tRNA Ser . After incubation at 37°C for 45 min, the reactions were quenched with ice-cold EDTA and spotted on PEI-cellulose TLC plates which were developed in 1 M LiCl for 60 to 75 min. PhosphorImager analysis was used to quantify the intensities of the [α- 32 P]ATP and [α- 32 P]ADP spots. The minimal ATPase activity in the absence of tRNA was subtracted. Error bars represent the standard deviation of three separate experiments.
Figure Legend Snippet: ATPase activity of M. jannaschii PSTK. Graph of the ratio of [α- 32 P]ATP converted to [α- 32 P]ADP by PSTK (200 nM) in the absence or presence of 1 µM tRNA Sec , Ser-tRNA Sec , or Ser-tRNA Ser . After incubation at 37°C for 45 min, the reactions were quenched with ice-cold EDTA and spotted on PEI-cellulose TLC plates which were developed in 1 M LiCl for 60 to 75 min. PhosphorImager analysis was used to quantify the intensities of the [α- 32 P]ATP and [α- 32 P]ADP spots. The minimal ATPase activity in the absence of tRNA was subtracted. Error bars represent the standard deviation of three separate experiments.

Techniques Used: Activity Assay, Size-exclusion Chromatography, Incubation, Thin Layer Chromatography, Standard Deviation

25) Product Images from "Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes"

Article Title: Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0409275102

Effect of down-regulation of REX1 expression on stability of the L-complex. ( A ) Equivalent amounts of clarified mitochondrial lysates from uninduced and tetracycline-induced REX1–RNAi cells were fractionated on 10–30% glycerol gradients, and the fractions were subjected to autoadenylation with [α- 32 P]ATP and electrophoresed in an 8–16% polyacrylamide SDS gel. The gel was blotted onto a membrane and exposed by using a PhosphorImager to visualize the endogenous REL1 and REL2 bands. ( B ) The blot was reacted with antibodies against MP81, MP63, and MP42. The REL2 and MP81 bands apparently released from the L-complex in the form of the REL2 subcomplex are indicated by circles in A and B , respectively. ( C ) In a separate experiment, the L-complex peak fraction from uninduced, 9-day RNAi-induced cells were autoadenylated, and equal amounts were separated in a denaturing gel.
Figure Legend Snippet: Effect of down-regulation of REX1 expression on stability of the L-complex. ( A ) Equivalent amounts of clarified mitochondrial lysates from uninduced and tetracycline-induced REX1–RNAi cells were fractionated on 10–30% glycerol gradients, and the fractions were subjected to autoadenylation with [α- 32 P]ATP and electrophoresed in an 8–16% polyacrylamide SDS gel. The gel was blotted onto a membrane and exposed by using a PhosphorImager to visualize the endogenous REL1 and REL2 bands. ( B ) The blot was reacted with antibodies against MP81, MP63, and MP42. The REL2 and MP81 bands apparently released from the L-complex in the form of the REL2 subcomplex are indicated by circles in A and B , respectively. ( C ) In a separate experiment, the L-complex peak fraction from uninduced, 9-day RNAi-induced cells were autoadenylated, and equal amounts were separated in a denaturing gel.

Techniques Used: Expressing, SDS-Gel

26) Product Images from "Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase"

Article Title: Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Journal: Journal of Virology

doi: 10.1128/JVI.75.17.8289-8297.2001

ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [α- 32 P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS–12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.
Figure Legend Snippet: ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [α- 32 P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS–12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Autoradiography

27) Product Images from "The RNA Polymerase of Influenza Virus, Bound to the 5? End of Virion RNA, Acts in cis To Polyadenylate mRNA"

Article Title: The RNA Polymerase of Influenza Virus, Bound to the 5? End of Virion RNA, Acts in cis To Polyadenylate mRNA

Journal: Journal of Virology

doi:

Effect of point mutations in the conserved sequence at the 5′ end of vRNA in the [α- 32 P]ATP incorporation assay. Except for the 2′ (G→U) vRNA mutant (lane 3), all vRNA templates were present in excess in
Figure Legend Snippet: Effect of point mutations in the conserved sequence at the 5′ end of vRNA in the [α- 32 P]ATP incorporation assay. Except for the 2′ (G→U) vRNA mutant (lane 3), all vRNA templates were present in excess in

Techniques Used: Sequencing, Mutagenesis

Quantitation of the effect of point mutations (1′ to 13′) in the 5′ end of the 49-mer vRNA-like template on polyadenylation activity assayed by the [α- 32 P]ATP incorporation assay. The mRNA products (high-molecular-weight
Figure Legend Snippet: Quantitation of the effect of point mutations (1′ to 13′) in the 5′ end of the 49-mer vRNA-like template on polyadenylation activity assayed by the [α- 32 P]ATP incorporation assay. The mRNA products (high-molecular-weight

Techniques Used: Quantitation Assay, Activity Assay, Molecular Weight

28) Product Images from "XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation"

Article Title: XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn957

XLF stimulates ligase IV re-adenylation following ligation. ( A ) LX was treated with pyrophosphate (PPi) for 15 min at room temperature. Following dialysis, 1 pmol LX was incubated with α- 32 P-ATP with or without 5 pmol XLF. XLF stimulated LX adenylation activity 2.5-fold. Results and error bars are the mean and SD of two experiments. ( B ) Following PPi treatment and dialysis, 1 pmol LX was incubated with α- 32 P-ATP, with or without 30 pmol XLF and 0.2 pmol DNA ends. ( C ) One picomole LX was incubated with 0.2 pmol DNA ends for 15 min to allow ligation and hence de-adenylation. α- 32 P-ATP was then added with or without 5 pmol XLF. XLF enhanced adenylation 3-fold. Whether XLF was present or absent during the pre-incubation step did not change the level of adenylation. Results are the mean and SD of two experiments. ( D ) Samples treated as in (C) were examined for ligation by gel electrophoresis. Ligation occurred during the preincubation step and peaked by 15 min. ( E ) Total 0.2 pmol LX were incubated with 0.8 pmol DNA ends with or without 1 pmol XLF without ATP for 15 min. Samples were examined for ligation. XLF did not impact upon the level of ligation. ( F ) Two milligrams WCE from human pre-B control cells (Nalm6) were immunoprecipitated with α-XRCC4 antibodies, split in half and washed either in buffer containing 1 or 0.12 M NaCl. Samples were treated with or without PPi in the presence or absence of ∼35 pmol DNA ends and were examined for adenylation activity. LX-αAMP is the adenylated LX complex. Immunoprecipitated samples were subjected to immunoblot analysis using α-DNA ligase IV antibodies. The graph shows arbitrary units of intensity of adenylation normalized to protein levels.
Figure Legend Snippet: XLF stimulates ligase IV re-adenylation following ligation. ( A ) LX was treated with pyrophosphate (PPi) for 15 min at room temperature. Following dialysis, 1 pmol LX was incubated with α- 32 P-ATP with or without 5 pmol XLF. XLF stimulated LX adenylation activity 2.5-fold. Results and error bars are the mean and SD of two experiments. ( B ) Following PPi treatment and dialysis, 1 pmol LX was incubated with α- 32 P-ATP, with or without 30 pmol XLF and 0.2 pmol DNA ends. ( C ) One picomole LX was incubated with 0.2 pmol DNA ends for 15 min to allow ligation and hence de-adenylation. α- 32 P-ATP was then added with or without 5 pmol XLF. XLF enhanced adenylation 3-fold. Whether XLF was present or absent during the pre-incubation step did not change the level of adenylation. Results are the mean and SD of two experiments. ( D ) Samples treated as in (C) were examined for ligation by gel electrophoresis. Ligation occurred during the preincubation step and peaked by 15 min. ( E ) Total 0.2 pmol LX were incubated with 0.8 pmol DNA ends with or without 1 pmol XLF without ATP for 15 min. Samples were examined for ligation. XLF did not impact upon the level of ligation. ( F ) Two milligrams WCE from human pre-B control cells (Nalm6) were immunoprecipitated with α-XRCC4 antibodies, split in half and washed either in buffer containing 1 or 0.12 M NaCl. Samples were treated with or without PPi in the presence or absence of ∼35 pmol DNA ends and were examined for adenylation activity. LX-αAMP is the adenylated LX complex. Immunoprecipitated samples were subjected to immunoblot analysis using α-DNA ligase IV antibodies. The graph shows arbitrary units of intensity of adenylation normalized to protein levels.

Techniques Used: Ligation, Incubation, Activity Assay, Nucleic Acid Electrophoresis, Immunoprecipitation

29) Product Images from "Vanillins--a novel family of DNA-PK inhibitors"

Article Title: Vanillins--a novel family of DNA-PK inhibitors

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkg753

Ligase adenylation and Ku-IP6 binding. ( a ) Ligase adenylation. (i) Incorporation of [α- 32 P]ATP by a purified Ligase I titration. (ii) Effect of vanillin on Ligase I at 190 ng. A weak signal was generated, therefore (iii) shows effect of vanillin titration on 760 ng Ligase I. (iv) Incorporation of [α- 32 P]ATP by GM00558 extract. (v) Ten microgram extract pre-incubated (10 min at 4°C) with vanillin. ( b ) Ku binding to IP6. Effect of vanillin on Ku binding to 3H-labelled IP6. Counts per minute (c.p.m.) of labelled IP6 eluted plotted against vanillin concentration.
Figure Legend Snippet: Ligase adenylation and Ku-IP6 binding. ( a ) Ligase adenylation. (i) Incorporation of [α- 32 P]ATP by a purified Ligase I titration. (ii) Effect of vanillin on Ligase I at 190 ng. A weak signal was generated, therefore (iii) shows effect of vanillin titration on 760 ng Ligase I. (iv) Incorporation of [α- 32 P]ATP by GM00558 extract. (v) Ten microgram extract pre-incubated (10 min at 4°C) with vanillin. ( b ) Ku binding to IP6. Effect of vanillin on Ku binding to 3H-labelled IP6. Counts per minute (c.p.m.) of labelled IP6 eluted plotted against vanillin concentration.

Techniques Used: Binding Assay, Purification, Titration, Generated, Incubation, Concentration Assay

30) Product Images from "RNAi is antagonized by A- > I hyper-editing"

Article Title: RNAi is antagonized by A- > I hyper-editing

Journal: EMBO Reports

doi: 10.1093/embo-reports/kve244

Fig. 2. A→I editing progressively inhibits RNAi and production of siRNAs. ( A ) CAT dsRNA was edited to varying degrees (5, 12, 19, 25 and 43% A→I) using ADAR2 and analysed by native gel electrophoresis. As the level of editing increased there was a corresponding reduction in their mobility on a native gel (for example, compare lanes 1 and 6). Each deaminated RNA was relatively homogeneous, as indicated by the relatively tight bands with lower mobility than unmodified dsRNA. ( B ) An RNAi assay was performed for 90 min where each CAT d-dsRNA was tested for its ability to activate RNAi (lanes 2–6) alongside dsRNA (lane 1) and ssRNA (lane 7). In this assay the control RNA used was ΔKP. As the level of editing increased in the d-dsRNAs, there was a clear reduction in the amount of RNAi (compare lanes 1 and 6). ( C ) The data from (B) are represented as the CAT/ΔKP ratio relative to the CAT/ΔKP ratio in the presence of ssRNA. In order to accurately quantitate the amount of target RNA remaining in the presence of highly edited d-dsRNA (lanes 4–6), it was necessary to determine the sum of the two upper bands that correspond to CAT RNA (marked with an asterisk in lane 6). The anomalous mobility of a small proportion of the CAT target RNA is the result of partial hybridization of the CAT target RNA with the 200-fold excess of unlabelled CAT d-dsRNA. An inverse correlation exists between the level of editing in d-dsRNA and its ability to induce RNAi. ( D ) The production of siRNAs from CAT dsRNA and d-dsRNAs was analysed using RNAs labelled with [α 32 P]ATP. This assay was performed alongside that shown in (B) using identical conditions. As the level of editing increased, the production of siRNAs showed a corresponding decrease (compare lanes 1 and 6). The percentage of the input RNA that was converted to siRNAs is shown below each lane.
Figure Legend Snippet: Fig. 2. A→I editing progressively inhibits RNAi and production of siRNAs. ( A ) CAT dsRNA was edited to varying degrees (5, 12, 19, 25 and 43% A→I) using ADAR2 and analysed by native gel electrophoresis. As the level of editing increased there was a corresponding reduction in their mobility on a native gel (for example, compare lanes 1 and 6). Each deaminated RNA was relatively homogeneous, as indicated by the relatively tight bands with lower mobility than unmodified dsRNA. ( B ) An RNAi assay was performed for 90 min where each CAT d-dsRNA was tested for its ability to activate RNAi (lanes 2–6) alongside dsRNA (lane 1) and ssRNA (lane 7). In this assay the control RNA used was ΔKP. As the level of editing increased in the d-dsRNAs, there was a clear reduction in the amount of RNAi (compare lanes 1 and 6). ( C ) The data from (B) are represented as the CAT/ΔKP ratio relative to the CAT/ΔKP ratio in the presence of ssRNA. In order to accurately quantitate the amount of target RNA remaining in the presence of highly edited d-dsRNA (lanes 4–6), it was necessary to determine the sum of the two upper bands that correspond to CAT RNA (marked with an asterisk in lane 6). The anomalous mobility of a small proportion of the CAT target RNA is the result of partial hybridization of the CAT target RNA with the 200-fold excess of unlabelled CAT d-dsRNA. An inverse correlation exists between the level of editing in d-dsRNA and its ability to induce RNAi. ( D ) The production of siRNAs from CAT dsRNA and d-dsRNAs was analysed using RNAs labelled with [α 32 P]ATP. This assay was performed alongside that shown in (B) using identical conditions. As the level of editing increased, the production of siRNAs showed a corresponding decrease (compare lanes 1 and 6). The percentage of the input RNA that was converted to siRNAs is shown below each lane.

Techniques Used: Nucleic Acid Electrophoresis, RNAi Assay, Hybridization

Related Articles

Clone Assay:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: The NTPase/helicase domain of NS3 (amino acids 182 to 619) and full-length NS5 were cloned into the pET-21a and pET-28a vectors, respectively, and expressed in Escherichia coli BL21 cells upon induction with isopropyl-β- d -thiogalactopyranoside at 30°C for 3 to 4 h. The recombinant NS5 and NS3 NTPase/helicase domains contained a His6 tag at the N and C termini, respectively, and were purified through a nickel column (Novagen, Madison, Wis.). .. The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: We used the plasmids carrying the sequences for the yeast tRNAPhe , tRNAAsp (wild type and deleted for stem-loop D), the P. abyssi tRNALeu/CAA , sR47, and the HIV TAR RNA cloned behind the T7 promoter to produce the corresponding RNA transcripts. .. Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix.

Centrifugation:

Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities
Article Snippet: .. HSPA8 (70 µg) was loaded with 100 µCi of α-32 P-ATP in a total volume of 120 µl of complex buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]–KOH, pH 7.5, 100 mM KCl, 11 mM MgOAc, and 25 µM ATP) for 30 min at 4°C, and HSPA8–32 P-ATP complex was obtained by centrifugation through a Microspin G-25 column (GE Healthcare, Chicago, IL). .. Labeled HSPA8 (7.8 μg) was incubated in the presence or absence of 5 μg of Sse1 or Sse1sbd at 30°C.

Amplification:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: The E. coli tRNAleu2 , wild type and C56U, C56G, G18C/G19C or G18A/G19A mutants were transcribed from PCR fragments obtained by amplification of E. coli genomic DNA with the oligonucleotide pairs formed with the EC Leu2(5′) and one of the EC Leu2 (3′) oligonucleotides. .. Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix.

Synthesized:

Article Title: Primase Directs the Release of DnaC from DnaB
Article Snippet: The 5′-biotinylated oligonucleotide for biosensor experiments was synthesized by an in-house facility. .. Commercial reagents were BSA, horseradish peroxidase conjugated to goat anti-rabbit IgG (Jackson Laboratories), ribo- and deoxyribonucleotides (Pharmacia P-L Biochemicals), [α-32 P] ATP (Amersham Biosciences), and [methyl-3 H] dTTP (MP Biochemicals).

Autoradiography:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: After addition of 7 μl of Stop solution, samples were analyzed by electrophoresis on 8 M urea/8% polyacrylamide gels and autoradiography. .. Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice.

Blocking Assay:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice. .. The minitray was placed on a 37°C metal heat block for 5 min and then on a Spectroline TM-312A UV Transilluminator (Spectronics, Westbury, NY) lid down with an ice pack on top.

Electrophoresis:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: After addition of 7 μl of Stop solution, samples were analyzed by electrophoresis on 8 M urea/8% polyacrylamide gels and autoradiography. .. Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice.

Incubation:

Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities
Article Snippet: HSPA8 (70 µg) was loaded with 100 µCi of α-32 P-ATP in a total volume of 120 µl of complex buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]–KOH, pH 7.5, 100 mM KCl, 11 mM MgOAc, and 25 µM ATP) for 30 min at 4°C, and HSPA8–32 P-ATP complex was obtained by centrifugation through a Microspin G-25 column (GE Healthcare, Chicago, IL). .. Labeled HSPA8 (7.8 μg) was incubated in the presence or absence of 5 μg of Sse1 or Sse1sbd at 30°C.

Article Title: Assays for transfer RNA-dependent amino acid biosynthesis
Article Snippet: .. In 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2 , 5 mM DTT and 50 μM NaPPi , 15 μM tRNA is incubated with the CCA-adding enzyme (50 μg/ml) and 1.6 μCi/μL of [α-32 P]ATP (GE Healthcare, PB10200) for 35 minutes at room temperature, normally in a reaction volume of 48 μL. ..

Article Title: Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity
Article Snippet: .. In a reaction volume of 25 μL, pure Hsc70 at a final concentration of 28 nM was incubated with 1 μCi (13 nM) [α-32 P]ATP (Amersham, Piscataway, NJ) and various amounts of pure SGT/UBP or bovine serum albumin (BSA). ..

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by addition of 1 μl of 0.5 M EDTA disodium salt.

Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I
Article Snippet: [α-32 P]ATP and [γ-32 P]ATP (3,000 Ci/mmol; 1 Ci = 37 GBq) were obtained from Amersham. .. Succinylacetone (Sigma) was dissolved at a concentration of 500 mM in 1 M Hepes, pH 7.5, then diluted to the appropriate concentrations in the incubation buffers.

Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme
Article Snippet: .. For AMP incorporation into mini-D73 N74 N75 , reaction mixtures containing 50 mM glycine-KOH, pH 8.5, 15 mM KCl, 10 mM MgCl2 , 10 mM β-mercaptoethanol, 100 μM ATP, 100 nM α-32 P ATP (3000 Ci/mmol; GE Healthcare), 5 μM mini-helix, and 1 μg/ml AFCCA were incubated at 45°C for 5 min. .. The assay procedures for CMP incorporation into mini-D73 N74 were the same as those described above, except that 100 μM CTP, 100 μM ATP, and 100 nM α-32 P CTP (3000 Ci/mmol; GE Healthcare) were used instead of the 100 μM ATP and 100 nM α-32 P ATP.

Activity Assay:

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate. .. The products of [α-32 P]ATP hydrolysis were analyzed by thin-layer chromatography (TLC) as per reference .

Expressing:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: Paragraph title: Expression, purification, and enzyme assays of the NTPase/helicase domain of NS3 and full-length NS5. ... The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

Generated:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: .. Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix. .. Cold or radioactive transcripts were purified by 8% polyacrylamide/urea gel electrophoresis, eluted in 500 mM NH4-acetate, 1 mM EDTA, and ethanol-precipitated in the presence of polyU.

other:

Article Title: Foot-and-Mouth Disease Virus Mutant with Decreased Sensitivity to Ribavirin: Implications for Error Catastrophe ▿
Article Snippet: The assay was performed with MOPS (morpholinepropanesulfonic acid; 30 mM, pH 7.0; Sigma), NaCl (33 mM), and Mg(CH3 COO)2 (15 mM) or MnCl2 (10 mM) with poly(rC) or poly(rU) (40 ng/μl), oligo(dG)15 or oligo(rA)6 (2.4 μM), [α-32 P]GTP or [α-32 P]ATP (20 mCi/mmol, 0.01 mCi/ml; Amersham), RTP (Moravek Biochemicals, Inc.), and 3D (3 μM) (in Tris-HCl [250 mM, pH 7.5], NaCl [250 mM], EDTA [1 mM], glycerol [10%, vol/vol]).

Sequencing:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: The sequence of the full-length template is: 3′- CTTCG AGG CGAAG AAAA C C T TC T TC TTTT CC T TCC T GGGG-5′ (hairpin inverted repeat and position + 1 are underlined, and the T residue replaced with an A residue at positions +3, +6, +9, +10, +11, +12, +15, or +19, in the variant templates are in bold). .. Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice.

Affinity Purification:

Article Title: Primase Directs the Release of DnaC from DnaB
Article Snippet: Commercial reagents were BSA, horseradish peroxidase conjugated to goat anti-rabbit IgG (Jackson Laboratories), ribo- and deoxyribonucleotides (Pharmacia P-L Biochemicals), [α-32 P] ATP (Amersham Biosciences), and [methyl-3 H] dTTP (MP Biochemicals). .. Affinity-purified rabbit polyclonal antibodies for DnaB and DnaC, and rabbit antiserum that specifically recognizes the C-terminal region of DnaA (amino acids 370-467) were prepared in our laboratory.

Recombinant:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: .. The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3. .. The reaction mixture was incubated at 37°C for 30 min, and the reaction was terminated by addition of 1 μl of 0.5 M EDTA disodium salt.

Nucleic Acid Electrophoresis:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix. .. Cold or radioactive transcripts were purified by 8% polyacrylamide/urea gel electrophoresis, eluted in 500 mM NH4-acetate, 1 mM EDTA, and ethanol-precipitated in the presence of polyU.

Mutagenesis:

Article Title: Primase Directs the Release of DnaC from DnaB
Article Snippet: Mutant DnaC proteins were purified as described in . .. Commercial reagents were BSA, horseradish peroxidase conjugated to goat anti-rabbit IgG (Jackson Laboratories), ribo- and deoxyribonucleotides (Pharmacia P-L Biochemicals), [α-32 P] ATP (Amersham Biosciences), and [methyl-3 H] dTTP (MP Biochemicals).

Nickel Column:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: The NTPase/helicase domain of NS3 (amino acids 182 to 619) and full-length NS5 were cloned into the pET-21a and pET-28a vectors, respectively, and expressed in Escherichia coli BL21 cells upon induction with isopropyl-β- d -thiogalactopyranoside at 30°C for 3 to 4 h. The recombinant NS5 and NS3 NTPase/helicase domains contained a His6 tag at the N and C termini, respectively, and were purified through a nickel column (Novagen, Madison, Wis.). .. The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

Labeling:

Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities
Article Snippet: HSPA8 (70 µg) was loaded with 100 µCi of α-32 P-ATP in a total volume of 120 µl of complex buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]–KOH, pH 7.5, 100 mM KCl, 11 mM MgOAc, and 25 µM ATP) for 30 min at 4°C, and HSPA8–32 P-ATP complex was obtained by centrifugation through a Microspin G-25 column (GE Healthcare, Chicago, IL). .. Labeled HSPA8 (7.8 μg) was incubated in the presence or absence of 5 μg of Sse1 or Sse1sbd at 30°C.

Article Title: Assays for transfer RNA-dependent amino acid biosynthesis
Article Snippet: In 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2 , 5 mM DTT and 50 μM NaPPi , 15 μM tRNA is incubated with the CCA-adding enzyme (50 μg/ml) and 1.6 μCi/μL of [α-32 P]ATP (GE Healthcare, PB10200) for 35 minutes at room temperature, normally in a reaction volume of 48 μL. .. Liquid scintillation counting can be used to determine the efficiency of labeling.

Purification:

Article Title: Assays for transfer RNA-dependent amino acid biosynthesis
Article Snippet: The 3′ terminal AMP of purified tRNA (tRNAGln or tRNAAsn ) is 32 P-labeled by using the Escherichia coli CCA-adding enzyme and [α-32 P]ATP (GE Healthcare, PB10200) [ ]. .. In 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2 , 5 mM DTT and 50 μM NaPPi , 15 μM tRNA is incubated with the CCA-adding enzyme (50 μg/ml) and 1.6 μCi/μL of [α-32 P]ATP (GE Healthcare, PB10200) for 35 minutes at room temperature, normally in a reaction volume of 48 μL.

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: Paragraph title: Expression, purification, and enzyme assays of the NTPase/helicase domain of NS3 and full-length NS5. ... The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

Article Title: Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination
Article Snippet: Radiolabeled α-[32 P]-ATP and γ-[32 P]-ATP were purchased from Amersham Bioscience. .. Bacteriophage M13mp18 circular ssDNA and supercoiled double-stranded DNA (dsDNA) replicative form I (RFI) were purified as described ( ).

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix. .. Cold or radioactive transcripts were purified by 8% polyacrylamide/urea gel electrophoresis, eluted in 500 mM NH4-acetate, 1 mM EDTA, and ethanol-precipitated in the presence of polyU.

Article Title: Primase Directs the Release of DnaC from DnaB
Article Snippet: Mutant DnaC proteins were purified as described in . .. Commercial reagents were BSA, horseradish peroxidase conjugated to goat anti-rabbit IgG (Jackson Laboratories), ribo- and deoxyribonucleotides (Pharmacia P-L Biochemicals), [α-32 P] ATP (Amersham Biosciences), and [methyl-3 H] dTTP (MP Biochemicals).

Polymerase Chain Reaction:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: The E. coli tRNAleu2 , wild type and C56U, C56G, G18C/G19C or G18A/G19A mutants were transcribed from PCR fragments obtained by amplification of E. coli genomic DNA with the oligonucleotide pairs formed with the EC Leu2(5′) and one of the EC Leu2 (3′) oligonucleotides. .. Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix.

Polyacrylamide Gel Electrophoresis:

Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme
Article Snippet: For AMP incorporation into mini-D73 N74 N75 , reaction mixtures containing 50 mM glycine-KOH, pH 8.5, 15 mM KCl, 10 mM MgCl2 , 10 mM β-mercaptoethanol, 100 μM ATP, 100 nM α-32 P ATP (3000 Ci/mmol; GE Healthcare), 5 μM mini-helix, and 1 μg/ml AFCCA were incubated at 45°C for 5 min. .. The products were separated by 12% (w/v) polyacrylamide gel electrophoresis under denaturing conditions, and the intensity of 32 P-labelled RNAs was quantified by a BAS-2500 imager (Fuji Film, Japan).

SDS Page:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice. .. After irradiation for 40 min at 312 nm , the samples were mixed with 5 μl of 2× loading buffer (100 mM Tris·HCl, pH 6.8/200 mM DTT/4% SDS/0.2% bromophenol blue/20% glycerol), heated at 96°C for 2 min, and analyzed by SDS/PAGE and phosphorimaging.

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: Aliquots of 10 μl were quenched with 5 μl of 3× SDS-EDTA loading dye (7.5% [wt/vol] SDS, 90 mM EDTA, 37.5 mM Tris-HCl [pH 6.8], 37.5% glycerol, 0.3 M dithiothreitol), loaded without prior heating on a 11% polyacrylamide gel, and separated by SDS-PAGE. .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate.

Software:

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: Gels were exposed to a PhosphorImager screen overnight, and images were detected on a Typhoon Trio PhosphorImager (Amersham Biosciences) and analyzed using the ImageQuant version 5.0 software (Molecular Dynamics). .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate.

Irradiation:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice. .. After irradiation for 40 min at 312 nm , the samples were mixed with 5 μl of 2× loading buffer (100 mM Tris·HCl, pH 6.8/200 mM DTT/4% SDS/0.2% bromophenol blue/20% glycerol), heated at 96°C for 2 min, and analyzed by SDS/PAGE and phosphorimaging.

Positron Emission Tomography:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: The NTPase/helicase domain of NS3 (amino acids 182 to 619) and full-length NS5 were cloned into the pET-21a and pET-28a vectors, respectively, and expressed in Escherichia coli BL21 cells upon induction with isopropyl-β- d -thiogalactopyranoside at 30°C for 3 to 4 h. The recombinant NS5 and NS3 NTPase/helicase domains contained a His6 tag at the N and C termini, respectively, and were purified through a nickel column (Novagen, Madison, Wis.). .. The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

In Vitro:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: Paragraph title: In vitro T7 transcription of tRNA genes ... Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix.

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate. .. The products of [α-32 P]ATP hydrolysis were analyzed by thin-layer chromatography (TLC) as per reference .

Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme
Article Snippet: Paragraph title: In vitro CMP and AMP incorporation assays ... For AMP incorporation into mini-D73 N74 N75 , reaction mixtures containing 50 mM glycine-KOH, pH 8.5, 15 mM KCl, 10 mM MgCl2 , 10 mM β-mercaptoethanol, 100 μM ATP, 100 nM α-32 P ATP (3000 Ci/mmol; GE Healthcare), 5 μM mini-helix, and 1 μg/ml AFCCA were incubated at 45°C for 5 min.

Phosphorylation Assay:

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate. .. The products of [α-32 P]ATP hydrolysis were analyzed by thin-layer chromatography (TLC) as per reference .

Concentration Assay:

Article Title: Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity
Article Snippet: .. In a reaction volume of 25 μL, pure Hsc70 at a final concentration of 28 nM was incubated with 1 μCi (13 nM) [α-32 P]ATP (Amersham, Piscataway, NJ) and various amounts of pure SGT/UBP or bovine serum albumin (BSA). ..

Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I
Article Snippet: [α-32 P]ATP and [γ-32 P]ATP (3,000 Ci/mmol; 1 Ci = 37 GBq) were obtained from Amersham. .. Succinylacetone (Sigma) was dissolved at a concentration of 500 mM in 1 M Hepes, pH 7.5, then diluted to the appropriate concentrations in the incubation buffers.

Thin Layer Chromatography:

Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination
Article Snippet: The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3. .. Baker, Phillipsburg, N.J.) and analyzed by ascending thin-layer chromatography using 0.375 M potassium phosphate as a running buffer (pH 3.5).

Article Title: Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination
Article Snippet: Radiolabeled α-[32 P]-ATP and γ-[32 P]-ATP were purchased from Amersham Bioscience. .. Polyethyleneimine (PEI)–cellulose thin layer chromatography (TLC) plates were purchased from EMD Chemicals.

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate. .. The products of [α-32 P]ATP hydrolysis were analyzed by thin-layer chromatography (TLC) as per reference .

E. coli Genomic Assay:

Article Title: The Cm56 tRNA modification in archaea is catalyzed either by a specific 2?-O-methylase, or a C/D sRNP
Article Snippet: The E. coli tRNAleu2 , wild type and C56U, C56G, G18C/G19C or G18A/G19A mutants were transcribed from PCR fragments obtained by amplification of E. coli genomic DNA with the oligonucleotide pairs formed with the EC Leu2(5′) and one of the EC Leu2 (3′) oligonucleotides. .. Radioactive (32 P) transcripts were generated using [α-32 P]ATP, [α-32 P]GTP, [α-32 P]CTP, or [α-32 P]UTP (20–50 μCi, 400–800 Ci/mM) (Amersham) in the transcription mix.

Staining:

Article Title: EspA, an Orphan Hybrid Histidine Protein Kinase, Regulates the Timing of Expression of Key Developmental Proteins of Myxococcus xanthus
Article Snippet: Gels were subsequently stained by Coomassie dye to detect protein. .. ATPase activity was measured under the conditions of the in vitro phosphorylation assay, except that 0.5 mM [α-32 P]ATP (110 TBq mmol−1 ; Amersham) was used as a substrate.

Variant Assay:

Article Title: Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Article Snippet: The sequence of the full-length template is: 3′- CTTCG AGG CGAAG AAAA C C T TC T TC TTTT CC T TCC T GGGG-5′ (hairpin inverted repeat and position + 1 are underlined, and the T residue replaced with an A residue at positions +3, +6, +9, +10, +11, +12, +15, or +19, in the variant templates are in bold). .. Then 1 μM N-terminally His-6-tagged mini-vRNAP, 1 μM DNA oligonucleotide, 2 μM Eco SSB, 0.5 mM 5-IodoUTP (Sigma), 1 mM GTP, 0.1 mM ATP, 2 μCi of [α-32 P]ATP (3,000 Ci/mmol, Amersham Pharmacia), and buffer A were mixed in a total volume of 5 μl in a 15-μl well of a polystyrene MicroWell minitray with lid (Nunc) on ice.

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    GE Healthcare α 32 p atp
    A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled <t>ATP-FAM.</t> (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.
    α 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled ATP-FAM. (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.

    Journal: Molecular Biology of the Cell

    Article Title: Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities

    doi: 10.1091/mbc.E17-01-0070

    Figure Lengend Snippet: A novel SBD mutant exhibits impaired chaperone holdase activity but retains Hsp70 nucleotide exchange capacity. (A) Crystal structure of the Sse1 β-domain, with amino acids selected for mutations highlighted in red ( Xu et al. , 2012 ). (B) Fluorescence anisotropy was performed with increasing concentrations of chaperone (Sse1 or Sse1 sbd ) binding fluorescently labeled ATP-FAM. (C) Nucleotide exchange activity assays using HSPA8 (Hsp70) prebound to α- 32 P-ATP in the presence or absence of Sse1. (D) Holdase experiments were conducted using chemically denatured FFL (200 nM) diluted into refolding buffer without chaperone (no chap), with Sse1 (400 nM), or with Sse1 sbd (400 nM). FFL diluted into denaturing buffer instead of folding buffer was used as an aggregation control (denat). (E) Differential centrifugation analysis of FFL aggregation in the absence of chaperone or with Sse1 or Sse1 sbd after a 30-min holdase assay. Samples were visualized by SDS–PAGE, followed by Coomassie stain, and scanning densitometry quantitation was performed to determine FFL aggregation under each condition. (F) Endpoint analysis of holdase experiments performed as in D, using denatured FFL with varying ratios of chaperone to substrate, quantified as fraction of total aggregation.

    Article Snippet: HSPA8 (70 µg) was loaded with 100 µCi of α-32 P-ATP in a total volume of 120 µl of complex buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES]–KOH, pH 7.5, 100 mM KCl, 11 mM MgOAc, and 25 µM ATP) for 30 min at 4°C, and HSPA8–32 P-ATP complex was obtained by centrifugation through a Microspin G-25 column (GE Healthcare, Chicago, IL).

    Techniques: Mutagenesis, Activity Assay, Fluorescence, Binding Assay, Labeling, Centrifugation, SDS Page, Staining, Quantitation Assay

    [ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-

    Journal: Methods (San Diego, Calif.)

    Article Title: Assays for transfer RNA-dependent amino acid biosynthesis

    doi: 10.1016/j.ymeth.2007.06.010

    Figure Lengend Snippet: [ 32 P]tRNA/Nuclease P1 amidotransferase assay for Gln-tRNA Gln formation. (I) The 3′ terminal AMP of tRNA Gln is 32 P-labeled by using the E. coli CCA-adding enzyme and [α- 32 P]ATP. The stars denote the radioactive label. Excess [α-

    Article Snippet: In 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2 , 5 mM DTT and 50 μM NaPPi , 15 μM tRNA is incubated with the CCA-adding enzyme (50 μg/ml) and 1.6 μCi/μL of [α-32 P]ATP (GE Healthcare, PB10200) for 35 minutes at room temperature, normally in a reaction volume of 48 μL.

    Techniques: Labeling

    CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Journal:

    Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

    doi: 10.1038/emboj.2008.124

    Figure Lengend Snippet: CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Article Snippet: For AMP incorporation into mini-D73 N74 N75 , reaction mixtures containing 50 mM glycine-KOH, pH 8.5, 15 mM KCl, 10 mM MgCl2 , 10 mM β-mercaptoethanol, 100 μM ATP, 100 nM α-32 P ATP (3000 Ci/mmol; GE Healthcare), 5 μM mini-helix, and 1 μg/ml AFCCA were incubated at 45°C for 5 min.

    Techniques: Mutagenesis

    Comparisons of promoter strength and structure. (A). Sequences and structures of the 65-bp constructs contain a linear promoter and a pre-melted region with or without 14S rRNA promoter. (B-D). transcripts yielded from the ab0ove constructs using Rpo41 wild-type or ΔN160 (0.5 μM) in the absence or presence of Mtf1 (2.5 μM), labeled with [γ- 32 P]GTP, [γ- 32 P]ATP, and [α- 32 P]ATP, respectively.

    Journal: PLoS ONE

    Article Title: Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41

    doi: 10.1371/journal.pone.0136879

    Figure Lengend Snippet: Comparisons of promoter strength and structure. (A). Sequences and structures of the 65-bp constructs contain a linear promoter and a pre-melted region with or without 14S rRNA promoter. (B-D). transcripts yielded from the ab0ove constructs using Rpo41 wild-type or ΔN160 (0.5 μM) in the absence or presence of Mtf1 (2.5 μM), labeled with [γ- 32 P]GTP, [γ- 32 P]ATP, and [α- 32 P]ATP, respectively.

    Article Snippet: Various labeled nucleotide [γ-32 P]ATP, [γ-32 P]GTP, or [α-32 P]ATP (GE Healthcare) was used in reactions as indicated.

    Techniques: Construct, Labeling

    Activities of Rpo41 variants on pre-melted promoter-containing DNA. (A). The duplex DNA template contains a 14S rRNA promoter, with non-complementary sequences from positions-4 to +2. (B) (1) [γ- 32 P]ATP labeled transcripts on the above template (1 μM) using Rpo41 (0.5 μM wild-type or a variant) in the absence or presence of Mtf1 (2.5 μM) were visualized on a polyacrylamide denaturing gel. (2) Same as (1), except that transcripts are labeled with [γ- 32 P]ATP, [γ- 32 P]GTP or [α- 32 P]ATP. C) The intensities of run-off transcripts in B (1) quantified using Quantity One software.

    Journal: PLoS ONE

    Article Title: Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41

    doi: 10.1371/journal.pone.0136879

    Figure Lengend Snippet: Activities of Rpo41 variants on pre-melted promoter-containing DNA. (A). The duplex DNA template contains a 14S rRNA promoter, with non-complementary sequences from positions-4 to +2. (B) (1) [γ- 32 P]ATP labeled transcripts on the above template (1 μM) using Rpo41 (0.5 μM wild-type or a variant) in the absence or presence of Mtf1 (2.5 μM) were visualized on a polyacrylamide denaturing gel. (2) Same as (1), except that transcripts are labeled with [γ- 32 P]ATP, [γ- 32 P]GTP or [α- 32 P]ATP. C) The intensities of run-off transcripts in B (1) quantified using Quantity One software.

    Article Snippet: Various labeled nucleotide [γ-32 P]ATP, [γ-32 P]GTP, or [α-32 P]ATP (GE Healthcare) was used in reactions as indicated.

    Techniques: Labeling, Variant Assay, Software