Structured Review

HARTMANN ANALYTIC α 32 p atp
Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] <t>ATP</t> was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.
α 32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/α 32 p atp/product/HARTMANN ANALYTIC
Average 92 stars, based on 8 article reviews
Price from $9.99 to $1999.99
α 32 p atp - by Bioz Stars, 2020-07
92/100 stars

Images

1) Product Images from "Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus"

Article Title: Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus

Journal: RNA Biology

doi: 10.1080/15476286.2019.1582953

Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] ATP was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.
Figure Legend Snippet: Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] ATP was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.

Techniques Used: Activity Assay, Incubation, Purification, Marker

2) Product Images from "Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells"

Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-12-28

AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.
Figure Legend Snippet: AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography

Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.
Figure Legend Snippet: Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.

Techniques Used: Labeling, Incubation

Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.
Figure Legend Snippet: Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography

3) Product Images from "Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells"

Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-12-28

AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.
Figure Legend Snippet: AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography

Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.
Figure Legend Snippet: Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.

Techniques Used: Labeling, Incubation

Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.
Figure Legend Snippet: Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography

4) Product Images from "2?-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family"

Article Title: 2?-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr038

hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32 P-labeled cap01 structure ( A ) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32 P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α- 32 P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, ( B ) or presence ( C ) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described ( 44 ). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m 6 A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32 P-labeled cap0 ( D ) or capG ( E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32 P-labeled phosphates.
Figure Legend Snippet: hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32 P-labeled cap01 structure ( A ) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32 P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α- 32 P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, ( B ) or presence ( C ) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described ( 44 ). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m 6 A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32 P-labeled cap0 ( D ) or capG ( E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32 P-labeled phosphates.

Techniques Used: Activity Assay, In Vitro, Labeling, Incubation, Purification, Autoradiography, Negative Control, Modification, Ethanol Precipitation

5) Product Images from "Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification"

Article Title: Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

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

doi: 10.1073/pnas.1516930113

NmFic E186G adenylylates GyrB. Autoradiographs obtained after incubation of NmFic wt ( Left ) and inhibition-relieved variant NmFic E186G ) ( Right ) with 40 nM α-[ 32 P]ATP, 15 mM MgCl 2 , and E. coli cell lysates overexpressing potential targets as indicated. Incubation was for 1 h at 30 °C.
Figure Legend Snippet: NmFic E186G adenylylates GyrB. Autoradiographs obtained after incubation of NmFic wt ( Left ) and inhibition-relieved variant NmFic E186G ) ( Right ) with 40 nM α-[ 32 P]ATP, 15 mM MgCl 2 , and E. coli cell lysates overexpressing potential targets as indicated. Incubation was for 1 h at 30 °C.

Techniques Used: Incubation, Inhibition, Variant Assay

6) Product Images from "Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold"

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064901

AMP transfer catalyzed by Fic proteins and their inhibition-relieved variants. Autoradiography of VbhA/VbhT(FIC), SoFic and NmFic (wt, wild type; E/G, E- > G mutant) after incubation with radioactively labeled α- 32 P-ATP.
Figure Legend Snippet: AMP transfer catalyzed by Fic proteins and their inhibition-relieved variants. Autoradiography of VbhA/VbhT(FIC), SoFic and NmFic (wt, wild type; E/G, E- > G mutant) after incubation with radioactively labeled α- 32 P-ATP.

Techniques Used: Inhibition, Autoradiography, Mutagenesis, Incubation, Labeling

Related Articles

In Vitro:

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold
Article Snippet: .. In vitro Adenylylation Assay Adenylylation activity of VbhA/VbhT(FIC), SoFic and NmFic constructs was assessed by incubating 125 ng, 1.25 µg and 2.5 µg of purified protein, respectively, with 10 µCi α-32 P-ATP (Hartmann Analytic) in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EGTA, 15 mM MgCl2 , and protease inhibitor cocktail (Roche). ..

Protease Inhibitor:

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold
Article Snippet: .. In vitro Adenylylation Assay Adenylylation activity of VbhA/VbhT(FIC), SoFic and NmFic constructs was assessed by incubating 125 ng, 1.25 µg and 2.5 µg of purified protein, respectively, with 10 µCi α-32 P-ATP (Hartmann Analytic) in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EGTA, 15 mM MgCl2 , and protease inhibitor cocktail (Roche). ..

Construct:

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold
Article Snippet: .. In vitro Adenylylation Assay Adenylylation activity of VbhA/VbhT(FIC), SoFic and NmFic constructs was assessed by incubating 125 ng, 1.25 µg and 2.5 µg of purified protein, respectively, with 10 µCi α-32 P-ATP (Hartmann Analytic) in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EGTA, 15 mM MgCl2 , and protease inhibitor cocktail (Roche). ..

Purification:

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold
Article Snippet: .. In vitro Adenylylation Assay Adenylylation activity of VbhA/VbhT(FIC), SoFic and NmFic constructs was assessed by incubating 125 ng, 1.25 µg and 2.5 µg of purified protein, respectively, with 10 µCi α-32 P-ATP (Hartmann Analytic) in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EGTA, 15 mM MgCl2 , and protease inhibitor cocktail (Roche). ..

Incubation:

Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells
Article Snippet: .. Labeling of VEGF-A165 with [γ-32 P]ATP and [α-32 P]ATP For labeling, 3 μg VEGF-A165 (unless otherwise noted) was incubated with 5 μCi each of [γ-32 P]ATP or [α-32 P]ATP (Hartmann Analytic, Braunschweig, Germany) and combined with 0.01 mM non-radioactive ATP (optionally containing 0.1 mM MgCl2 ). .. Incubation was performed in 25 mM Tris-HCl (pH 7.5, total volume 15 μL, 37°C, 15 min).

Activity Assay:

Article Title: Conserved Inhibitory Mechanism and Competent ATP Binding Mode for Adenylyltransferases with Fic Fold
Article Snippet: .. In vitro Adenylylation Assay Adenylylation activity of VbhA/VbhT(FIC), SoFic and NmFic constructs was assessed by incubating 125 ng, 1.25 µg and 2.5 µg of purified protein, respectively, with 10 µCi α-32 P-ATP (Hartmann Analytic) in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM EGTA, 15 mM MgCl2 , and protease inhibitor cocktail (Roche). ..

Article Title: Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis *
Article Snippet: .. [α-32 P]ATP was obtained from Hartmann Analytic (Braunschweig, Germany) at a specific activity of 15 TBq (400 Ci)/mmol. .. Polyethyleneimine TLC plates were provided by VWR International.

Article Title: Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification
Article Snippet: .. Adenylylation activity of NmFic was assessed by incubating NmFic with 5 mM ATP, 10 μCi [α-32 P]-ATP (Hartmann Analytic), 25 mM MgCl2 ). .. NmFicE156R and NmFicE156R,Y183F (10 mg/mL and 8 mg/mL, respectively) crystallized in 500 μL of precipitant solution containing 10 mM Tris (pH 7.8) and 100 mM NaCl using the batch crystallization method at 4 °C.

Labeling:

Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells
Article Snippet: .. Labeling of VEGF-A165 with [γ-32 P]ATP and [α-32 P]ATP For labeling, 3 μg VEGF-A165 (unless otherwise noted) was incubated with 5 μCi each of [γ-32 P]ATP or [α-32 P]ATP (Hartmann Analytic, Braunschweig, Germany) and combined with 0.01 mM non-radioactive ATP (optionally containing 0.1 mM MgCl2 ). .. Incubation was performed in 25 mM Tris-HCl (pH 7.5, total volume 15 μL, 37°C, 15 min).

End Labeling:

Article Title: RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth
Article Snippet: .. Oligodeoxynucleotides used for hybridization are listed in Table S1. α-32 [P]-ATP (SRP-301; Hartmann Analytic) and T4 polynucleotide kinase (#EK0031; Fermentas) were used for the end-labeling reaction. .. For detection of SorX, a PCR product (primers listed in Table S1) was labeled with α-[32 P]-dCTP (SRP-205; Hartmann Analytic) using the Prime-a-Gene Labeling System (U1100; Promega) as described in the manufacturer’s manual.

Hybridization:

Article Title: RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth
Article Snippet: .. Oligodeoxynucleotides used for hybridization are listed in Table S1. α-32 [P]-ATP (SRP-301; Hartmann Analytic) and T4 polynucleotide kinase (#EK0031; Fermentas) were used for the end-labeling reaction. .. For detection of SorX, a PCR product (primers listed in Table S1) was labeled with α-[32 P]-dCTP (SRP-205; Hartmann Analytic) using the Prime-a-Gene Labeling System (U1100; Promega) as described in the manufacturer’s manual.

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    HARTMANN ANALYTIC α 32 p atp
    Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] <t>ATP</t> was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.
    α 32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p atp/product/HARTMANN ANALYTIC
    Average 92 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    α 32 p atp - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] ATP was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.

    Journal: RNA Biology

    Article Title: Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus

    doi: 10.1080/15476286.2019.1582953

    Figure Lengend Snippet: Endoribonucleolytic activity of Sso aIF5A. (a) Lane 1, [α- 32 P] ATP was loaded on the gel. Lane 2, incubation of 5′-PPP-GGA*-″-3′ RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5′-PPP-GGA*-″-3′ RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The blue arrows indicate the full-length RNA and the single A nucleotide. The red arrow indicates a stable degradation product. (b) Lane 1, RNA size marker. Lane 2, incubation of 5´end-labelled 2508sh RNA for 22 min at 65°C. Lanes 3–14, time course of degradation of 5´end-labelled 2508sh RNA at 65°C in the presence of N-His-aIF5A purified from E. coli . The arrow indicates the accumulation of a stable degradation product. Only the relevant part of the autoradiogram is shown.

    Article Snippet: 10 pmol of dephosphorylated 2508sh RNA were radiolabeled at the 5´-end with T4 Polynucleotide Kinase (Thermo Scientific) in the presence of 15 pmol of [α-32 P]ATP (3000 Ci/mmol, Hartmann Analytic GmbH).

    Techniques: Activity Assay, Incubation, Purification, Marker

    AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.

    Journal: BMC Biochemistry

    Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

    doi: 10.1186/1471-2091-12-28

    Figure Lengend Snippet: AP hydrolyzes [γ- 32 P]ATP and [α- 32 P]ATP bound to VEGF-A 165 . VEGF-A 165 (15 μM) was labeled with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 15 min. After labeling (t = 15 min), 300 ng of AP was added (lane 3 and 4). Incubation of all samples was continued for an additional period of 15 min followed by SDS-PAGE and autoradiography.

    Article Snippet: Labeling of VEGF-A165 with [γ-32 P]ATP and [α-32 P]ATP For labeling, 3 μg VEGF-A165 (unless otherwise noted) was incubated with 5 μCi each of [γ-32 P]ATP or [α-32 P]ATP (Hartmann Analytic, Braunschweig, Germany) and combined with 0.01 mM non-radioactive ATP (optionally containing 0.1 mM MgCl2 ).

    Techniques: Labeling, Incubation, SDS Page, Autoradiography

    Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.

    Journal: BMC Biochemistry

    Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

    doi: 10.1186/1471-2091-12-28

    Figure Lengend Snippet: Effect of increased ionic strength on labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (3 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C for 30 min. NaCl (100 mM) was added at times (t) indicated.

    Article Snippet: Labeling of VEGF-A165 with [γ-32 P]ATP and [α-32 P]ATP For labeling, 3 μg VEGF-A165 (unless otherwise noted) was incubated with 5 μCi each of [γ-32 P]ATP or [α-32 P]ATP (Hartmann Analytic, Braunschweig, Germany) and combined with 0.01 mM non-radioactive ATP (optionally containing 0.1 mM MgCl2 ).

    Techniques: Labeling, Incubation

    Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.

    Journal: BMC Biochemistry

    Article Title: Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

    doi: 10.1186/1471-2091-12-28

    Figure Lengend Snippet: Labeling of VEGF-A 165 with [γ- 32 P]ATP and [α- 32 P]ATP . VEGF-A 165 (2 μg) was incubated with radioactive ATP (5 μCi) in Tris-HCl (pH 7.5) at 37°C. MgCl 2 (0.1 mM) was added prior to ATP (+). After 15 min of incubation SDS-PAGE and autoradiography were performed.

    Article Snippet: Labeling of VEGF-A165 with [γ-32 P]ATP and [α-32 P]ATP For labeling, 3 μg VEGF-A165 (unless otherwise noted) was incubated with 5 μCi each of [γ-32 P]ATP or [α-32 P]ATP (Hartmann Analytic, Braunschweig, Germany) and combined with 0.01 mM non-radioactive ATP (optionally containing 0.1 mM MgCl2 ).

    Techniques: Labeling, Incubation, SDS Page, Autoradiography

    hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32 P-labeled cap01 structure ( A ) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32 P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α- 32 P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, ( B ) or presence ( C ) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described ( 44 ). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m 6 A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32 P-labeled cap0 ( D ) or capG ( E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32 P-labeled phosphates.

    Journal: Nucleic Acids Research

    Article Title: 2?-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family

    doi: 10.1093/nar/gkr038

    Figure Lengend Snippet: hMTr2 activity and substrate requirements. Methyltransferase activity: In vitro transcribed RNA-GG molecules with the 32 P-labeled cap01 structure ( A ) were incubated with enzymes as indicated in the presence of SAM. Purified product RNA was digested with RNase T2. Digestion products were resolved on 21% polyacrylamide/8 M urea gel and visualized by autoradiography. BAP protein was used as negative control. RNA with 32 P-labeled cap structure created with the TbMTr2 enzyme was used as a reference. Specificity: autoradiography of two-dimensional chromatograms of 5′-phosphate nucleosides on thin layer cellulose plates. [α- 32 P] ATP-labeled in vitro transcribed cap01-RNA-GA was incubated with SAM in the absence, ( B ) or presence ( C ) of the hMTr2 protein. Product RNA was purified, cleaved by nuclease P1 and the resulting nucleotides were analyzed as described ( 44 ). 5′-monophosphate ribonucleosides of G, A, U, C, Am and m 6 A were used as standards. Substrate requirements: In vitro transcribed RNA-GG molecules with 32 P-labeled cap0 ( D ) or capG ( E) structure were incubated with one of the indicated enzymes, added in a given order, in the presence of SAM. After every modification step RNA molecules were purified by phenol/chloroform extraction and ethanol precipitation. Final products were digested and analyzed as described in legend for panel A. Asterisks indicate positions of 32 P-labeled phosphates.

    Article Snippet: For generating internally, 32 P-labeled G-capped transcript RNA-GA [Gppp G p* A pGp(TpCp)12 ], transcription was carried out using pTZ19R-RNA-GA template, prepared as described above, with the addition of 10 μCi of [α-32 P] ATP (3000 Ci/mmol; Hartman Analytic GmbH) and the GpppG cap analog (Epicentre) following the manufacturer instructions.

    Techniques: Activity Assay, In Vitro, Labeling, Incubation, Purification, Autoradiography, Negative Control, Modification, Ethanol Precipitation