Structured Review

PerkinElmer mbq α 32 p atp
FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 <t>P-ATP.</t> A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .
Mbq α 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP"

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.3337

FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 P-ATP. A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .
Figure Legend Snippet: FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 P-ATP. A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .

Techniques Used: In Vitro, Autoradiography, SDS Page, Mutagenesis, Electrofocusing, Western Blot, Staining, Fluorescence, Labeling

2) Product Images from "AMPylation matches BiP activity to client protein load in the endoplasmic reticulum"

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

Journal: eLife

doi: 10.7554/eLife.12621

Mutation of threonine 518 in the substrate binding domain of BiP abolishes its AMPylation in vitro. ( A ) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant bacterially-expressed wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICD E234G coupled to GSH-Sepharose beads (lanes 2-5) or GST alone as a control (lane 1) in the presence of α- 32 P-ATP as a substrate. ( B ) Coomassie-stained native gel of wildtype BiP and the indicated mutants (all at 20 µM), following exposure to ATP (1.5 mM), GST-FICD E234G (0.8 µM), both or neither (for 45 min at 30°C). Where indicated the samples were afterwards exposed to SubA (30 ng/µl, 10 min at room temperature). ( C ) As in “A” above, with a different set of mutant BiP proteins. ( D ) As in “B” above, with a different set of mutant BiP proteins. Note that both the T518E and T518A mutations (in panel “B” above) affect the mobility of the ‘A’ form of BiP and forestall further changes in mobility by FICD, but only the T518E mutation mimics enzyme-mediated AMPylation by promoting a ‘B’ form-like state partially resistant to cleavage by SubA. DOI: http://dx.doi.org/10.7554/eLife.12621.012
Figure Legend Snippet: Mutation of threonine 518 in the substrate binding domain of BiP abolishes its AMPylation in vitro. ( A ) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant bacterially-expressed wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICD E234G coupled to GSH-Sepharose beads (lanes 2-5) or GST alone as a control (lane 1) in the presence of α- 32 P-ATP as a substrate. ( B ) Coomassie-stained native gel of wildtype BiP and the indicated mutants (all at 20 µM), following exposure to ATP (1.5 mM), GST-FICD E234G (0.8 µM), both or neither (for 45 min at 30°C). Where indicated the samples were afterwards exposed to SubA (30 ng/µl, 10 min at room temperature). ( C ) As in “A” above, with a different set of mutant BiP proteins. ( D ) As in “B” above, with a different set of mutant BiP proteins. Note that both the T518E and T518A mutations (in panel “B” above) affect the mobility of the ‘A’ form of BiP and forestall further changes in mobility by FICD, but only the T518E mutation mimics enzyme-mediated AMPylation by promoting a ‘B’ form-like state partially resistant to cleavage by SubA. DOI: http://dx.doi.org/10.7554/eLife.12621.012

Techniques Used: Mutagenesis, Binding Assay, In Vitro, Autoradiography, Staining, SDS Page, Recombinant

The isolated BiP substrate binding domain is not measurably AMPylated by FICD in vitro. Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and a fusion of the isolated substrate binding domain (SBD) to Smt3 (Smt3-SBD) following exposure in vitro to active GST-FICD E243G coupled to GSH-Sepharose beads (lanes 2-3) or GST alone as a control, (lane 1) in the presence of α- 32 P-ATP as a substrate. DOI: http://dx.doi.org/10.7554/eLife.12621.017
Figure Legend Snippet: The isolated BiP substrate binding domain is not measurably AMPylated by FICD in vitro. Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and a fusion of the isolated substrate binding domain (SBD) to Smt3 (Smt3-SBD) following exposure in vitro to active GST-FICD E243G coupled to GSH-Sepharose beads (lanes 2-3) or GST alone as a control, (lane 1) in the presence of α- 32 P-ATP as a substrate. DOI: http://dx.doi.org/10.7554/eLife.12621.017

Techniques Used: Isolation, Binding Assay, In Vitro, Autoradiography, Staining, SDS Page

Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro. (A) Schema of the experimental design. ATP hydrolysis-deficient BiP T229A protein was AMPylated in presence of radioactive α- 32 P-ATP with catalytically active GST-FICD E234G coupled to GSH-Sepharose beads. The enzyme-containing beads were removed by centrifugation and excess of non-radioactive ATP was added to the supernatant to competitively displace non-covalently bound α- 32 P-ATP from BiP T229A . Unbound nucleotides were then removed by passing the sample through a desalting column. Trace amounts (1.5 µg) of the recovered 32 P-labeled AMPylated BiP T229A protein were added to excess of unmodified BiP T229A protein (60 µg; a mass ratio of 40:1) and the combined sample was supplemented with ATP and treated for 30 min with increasing concentrations of the SubA protease (0.08 to 120 ng/µl) before denaturing SDS-PAGE, Coomassie staining and autoradiography. ( B ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples described above. In the bottom panel, the Coomassie stain and radioactive signals of the full-length BiP and the substrate binding domain (SBD) were quantified and normalized to the values in lane 1, which were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain signal reports of the fate of unmodified BiP T229A , whereas the radioactive signals report exclusively on the modified BiP T229A in the combined sample. NBD denotes the nucleotide binding domain. ( C ) A shorter exposure (1 hr versus 8 hr) of the autoradiograph shown in “B”, above. It reveals the substantial conservation of the radioactive signal emanating from intact BiP across the time course (the green plot in “B” above), which is obscured by changes in the band width introduced by the progressive digestion of the unlabeled (and unmodified) intact BiP in the sample. DOI: http://dx.doi.org/10.7554/eLife.12621.007
Figure Legend Snippet: Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro. (A) Schema of the experimental design. ATP hydrolysis-deficient BiP T229A protein was AMPylated in presence of radioactive α- 32 P-ATP with catalytically active GST-FICD E234G coupled to GSH-Sepharose beads. The enzyme-containing beads were removed by centrifugation and excess of non-radioactive ATP was added to the supernatant to competitively displace non-covalently bound α- 32 P-ATP from BiP T229A . Unbound nucleotides were then removed by passing the sample through a desalting column. Trace amounts (1.5 µg) of the recovered 32 P-labeled AMPylated BiP T229A protein were added to excess of unmodified BiP T229A protein (60 µg; a mass ratio of 40:1) and the combined sample was supplemented with ATP and treated for 30 min with increasing concentrations of the SubA protease (0.08 to 120 ng/µl) before denaturing SDS-PAGE, Coomassie staining and autoradiography. ( B ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples described above. In the bottom panel, the Coomassie stain and radioactive signals of the full-length BiP and the substrate binding domain (SBD) were quantified and normalized to the values in lane 1, which were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain signal reports of the fate of unmodified BiP T229A , whereas the radioactive signals report exclusively on the modified BiP T229A in the combined sample. NBD denotes the nucleotide binding domain. ( C ) A shorter exposure (1 hr versus 8 hr) of the autoradiograph shown in “B”, above. It reveals the substantial conservation of the radioactive signal emanating from intact BiP across the time course (the green plot in “B” above), which is obscured by changes in the band width introduced by the progressive digestion of the unlabeled (and unmodified) intact BiP in the sample. DOI: http://dx.doi.org/10.7554/eLife.12621.007

Techniques Used: In Vitro, Centrifugation, Labeling, SDS Page, Staining, Autoradiography, Binding Assay, Modification

Related Articles

Centrifugation:

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer. .. After 45 min [in these experiments a time before the reaction went to completion to reveal differences in AMPylation amongst BiP mutants ( and )] the beads were sedimented by centrifugation and the reaction was terminated by addition of SDS loading buffer to the supernatants and heating for 5 min at 75°C.

Filtration:

Article Title: AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation
Article Snippet: Unmodified and AMPylated BiP proteins were adjusted to 30 µM in reaction solution (25 mM HEPES-KOH pH 7.4, 50 mM KCl, 10 mM MgCl2 ) and incubated in a final volume of 50 µl with 0.8 mM ATP and 0.6 MBq α-32 P-ATP (EasyTide; Perkin Elmer, Waltham, MA) for 3 min on ice. .. The formed BiP•ATP complexes were separated from free nucleotide by gel filtration using illustra Sephadex G-50 NICK columns (GE Healthcare, cat. no. 17-0855-01) that have been pre-saturated with 1 ml of a 1 mg/ml bovine serum albumin (BSA) solution and equilibrated with ice-cold reaction solution.

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: .. Single-turnover ATPase assay Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. ..

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: .. Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. ..

In Vitro:

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: .. Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer. .. The reactions were incubated with 5 µl GSH-Sepharose beads coupled with mammalian expressed GST-FICDE234G (or GST only) in 1.5 ml reaction tubes at 30°C on a thermomixer (Eppendorf) while shaking at 800 rpm.

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP
Article Snippet: .. Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer). .. After 3 and 10 minutes of incubation 15 µl were removed from each reaction, respectively, supplemented with 5 µl SDS sample buffer, heated for 5 minutes at 75°C and loaded on a SDS-PAGE gel.

Mutagenesis:

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: .. Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer. .. The reactions were incubated with 5 µl GSH-Sepharose beads coupled with mammalian expressed GST-FICDE234G (or GST only) in 1.5 ml reaction tubes at 30°C on a thermomixer (Eppendorf) while shaking at 800 rpm.

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP
Article Snippet: .. Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer). .. After 3 and 10 minutes of incubation 15 µl were removed from each reaction, respectively, supplemented with 5 µl SDS sample buffer, heated for 5 minutes at 75°C and loaded on a SDS-PAGE gel.

Isolation:

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: .. Single-turnover ATPase assay Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. ..

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: .. Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. ..

Purification:

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: .. Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer. .. The reactions were incubated with 5 µl GSH-Sepharose beads coupled with mammalian expressed GST-FICDE234G (or GST only) in 1.5 ml reaction tubes at 30°C on a thermomixer (Eppendorf) while shaking at 800 rpm.

Incubation:

Article Title: AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation
Article Snippet: .. Unmodified and AMPylated BiP proteins were adjusted to 30 µM in reaction solution (25 mM HEPES-KOH pH 7.4, 50 mM KCl, 10 mM MgCl2 ) and incubated in a final volume of 50 µl with 0.8 mM ATP and 0.6 MBq α-32 P-ATP (EasyTide; Perkin Elmer, Waltham, MA) for 3 min on ice. .. The formed BiP•ATP complexes were separated from free nucleotide by gel filtration using illustra Sephadex G-50 NICK columns (GE Healthcare, cat. no. 17-0855-01) that have been pre-saturated with 1 ml of a 1 mg/ml bovine serum albumin (BSA) solution and equilibrated with ice-cold reaction solution.

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: Single-turnover ATPase assay Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. .. The reactions were incubated at 30 °C and samples were taken at the indicated time points and directly spotted onto a thin-layer chromatography (TLC) plate.

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. .. The reactions were incubated at 30 °C and samples were taken at the indicated time points and directly spotted onto a thin-layer chromatography (TLC) plate.

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: The in vitro AMPylation time-course experiments presented in were performed likewise but after incubation for the indicated times at 30°C the reactions were stopped on ice by addition of 40 mM EDTA and samples were run immediately on native gels. .. Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer.

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP
Article Snippet: Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer). .. After 3 and 10 minutes of incubation 15 µl were removed from each reaction, respectively, supplemented with 5 µl SDS sample buffer, heated for 5 minutes at 75°C and loaded on a SDS-PAGE gel.

Thin Layer Chromatography:

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: Single-turnover ATPase assay Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. .. The reactions were incubated at 30 °C and samples were taken at the indicated time points and directly spotted onto a thin-layer chromatography (TLC) plate.

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. .. The reactions were incubated at 30 °C and samples were taken at the indicated time points and directly spotted onto a thin-layer chromatography (TLC) plate.

Staining:

Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum
Article Snippet: Volumes corresponding to 7 µg BiP protein were loaded per lane on native or SDS polyacrylamide gels and visualized by Coomassie staining. .. Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer.

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP
Article Snippet: Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer). .. Gels were stained with Coomassie and the radioactive signals were detected with a Typhoon Trio imager (GE Healthcare) upon overnight exposure of the dried gels to a storage phosphor screen.

ATPase Assay:

Article Title: AMPylation targets the rate-limiting step of BiP’s ATPase cycle for its functional inactivation
Article Snippet: Paragraph title: Single-turnover ATPase assay ... Unmodified and AMPylated BiP proteins were adjusted to 30 µM in reaction solution (25 mM HEPES-KOH pH 7.4, 50 mM KCl, 10 mM MgCl2 ) and incubated in a final volume of 50 µl with 0.8 mM ATP and 0.6 MBq α-32 P-ATP (EasyTide; Perkin Elmer, Waltham, MA) for 3 min on ice.

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: .. Single-turnover ATPase assay Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer. ..

Article Title: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
Article Snippet: Paragraph title: Single-turnover ATPase assay ... Single-turnover experiments were performed according to a previously described procedure as follows: complexes of BiP with ATP were formed in 50-µl reactions containing 30 µM wild-type BiP protein (UK173), 800 µM ATP, 0.444 MBq [α-32 P]-ATP (EasyTide; Perkin Elmer) in 25 mM HEPES–KOH pH 7.4, 100 mM KCl, 10 mM MgCl2 for 3 min on ice, and isolated by gel filtration using a illustra Sephadex G-50 NICK column (GE Healthcare) pre-saturated with 1 mg/ml BSA solution and equilibrated in reaction buffer.

SDS Page:

Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP
Article Snippet: Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer). .. After 3 and 10 minutes of incubation 15 µl were removed from each reaction, respectively, supplemented with 5 µl SDS sample buffer, heated for 5 minutes at 75°C and loaded on a SDS-PAGE gel.

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  • 94
    PerkinElmer mbq α 32 p atp
    FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 <t>P-ATP.</t> A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .
    Mbq α 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer α 32 p atp
    KaiA-stimulated nucleotide exchange on KaiC. ( A and B ) KaiC-bound radioactive nucleotides were chased with exogenous nonradioactive <t>ATP.</t> KaiC hexamers were prepared from monomers in the presence of 2 mM [α- 32 P]ATP, unbound nucleotides were removed,
    α 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p atp/product/PerkinElmer
    Average 95 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
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    FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 P-ATP. A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .

    Journal: Nature structural & molecular biology

    Article Title: FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

    doi: 10.1038/nsmb.3337

    Figure Lengend Snippet: FICD de-AMPylates BiP in vitro ( a ) Autoradiograph of an SDS-PAGE gel loaded with AMPylated BiP (BiP- 32 P-AMP) that had been exposed to wildtype of mutant FICD for the indicated time Wildtype FICD-dependent de-AMPylation of BiP- 32 P-AMP was observed in four independent experiments. ( b ) IEF immunoblots of endogenous BiP from lysates of untreated or cycloheximide-treated (CHX) CHO-K1 cells that had been reacted in vitro with the indicated FICD enzymes. ( c ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP after exposure to wildtype or mutant versions of FICD in presence of α- 32 P-ATP. A representative of three independent experiments is shown ( n = 3). ( d ) Time-dependent plot of fluorescence polarization (FP) of BiP AMPylated with FAM-labeled AMP (BiP T518-AMP-FAM ) following exposure to the indicated FICD proteins. The decrease in the FP signal reflects release of the fluorophore from BiP. A representative of five independent experiments is shown ( n = 5). Uncropped autoradiograph, gel and blot images are shown in Supplementary Data Set 1 .

    Article Snippet: Radioactive in vitro AMPylation ( ) reactions were set up in a final volume of 37.5 µl containing 1 µM of ATP hydrolysis-deficient mutant BiP protein (BiPT229A ) , 0.1 µM wildtype or mutant FICD proteins, 40 µM ATP, and 0.023 MBq α-32 P-ATP (EasyTide; Perkin Elmer).

    Techniques: In Vitro, Autoradiography, SDS Page, Mutagenesis, Electrofocusing, Western Blot, Staining, Fluorescence, Labeling

    Mutation of threonine 518 in the substrate binding domain of BiP abolishes its AMPylation in vitro. ( A ) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant bacterially-expressed wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICD E234G coupled to GSH-Sepharose beads (lanes 2-5) or GST alone as a control (lane 1) in the presence of α- 32 P-ATP as a substrate. ( B ) Coomassie-stained native gel of wildtype BiP and the indicated mutants (all at 20 µM), following exposure to ATP (1.5 mM), GST-FICD E234G (0.8 µM), both or neither (for 45 min at 30°C). Where indicated the samples were afterwards exposed to SubA (30 ng/µl, 10 min at room temperature). ( C ) As in “A” above, with a different set of mutant BiP proteins. ( D ) As in “B” above, with a different set of mutant BiP proteins. Note that both the T518E and T518A mutations (in panel “B” above) affect the mobility of the ‘A’ form of BiP and forestall further changes in mobility by FICD, but only the T518E mutation mimics enzyme-mediated AMPylation by promoting a ‘B’ form-like state partially resistant to cleavage by SubA. DOI: http://dx.doi.org/10.7554/eLife.12621.012

    Journal: eLife

    Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

    doi: 10.7554/eLife.12621

    Figure Lengend Snippet: Mutation of threonine 518 in the substrate binding domain of BiP abolishes its AMPylation in vitro. ( A ) Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of recombinant bacterially-expressed wildtype (wt) BiP and the indicated mutants exposed in vitro to active GST-FICD E234G coupled to GSH-Sepharose beads (lanes 2-5) or GST alone as a control (lane 1) in the presence of α- 32 P-ATP as a substrate. ( B ) Coomassie-stained native gel of wildtype BiP and the indicated mutants (all at 20 µM), following exposure to ATP (1.5 mM), GST-FICD E234G (0.8 µM), both or neither (for 45 min at 30°C). Where indicated the samples were afterwards exposed to SubA (30 ng/µl, 10 min at room temperature). ( C ) As in “A” above, with a different set of mutant BiP proteins. ( D ) As in “B” above, with a different set of mutant BiP proteins. Note that both the T518E and T518A mutations (in panel “B” above) affect the mobility of the ‘A’ form of BiP and forestall further changes in mobility by FICD, but only the T518E mutation mimics enzyme-mediated AMPylation by promoting a ‘B’ form-like state partially resistant to cleavage by SubA. DOI: http://dx.doi.org/10.7554/eLife.12621.012

    Article Snippet: Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer.

    Techniques: Mutagenesis, Binding Assay, In Vitro, Autoradiography, Staining, SDS Page, Recombinant

    The isolated BiP substrate binding domain is not measurably AMPylated by FICD in vitro. Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and a fusion of the isolated substrate binding domain (SBD) to Smt3 (Smt3-SBD) following exposure in vitro to active GST-FICD E243G coupled to GSH-Sepharose beads (lanes 2-3) or GST alone as a control, (lane 1) in the presence of α- 32 P-ATP as a substrate. DOI: http://dx.doi.org/10.7554/eLife.12621.017

    Journal: eLife

    Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

    doi: 10.7554/eLife.12621

    Figure Lengend Snippet: The isolated BiP substrate binding domain is not measurably AMPylated by FICD in vitro. Autoradiograph and Coomassie (CBB) stain of an SDS-PAGE gel of wildtype BiP and a fusion of the isolated substrate binding domain (SBD) to Smt3 (Smt3-SBD) following exposure in vitro to active GST-FICD E243G coupled to GSH-Sepharose beads (lanes 2-3) or GST alone as a control, (lane 1) in the presence of α- 32 P-ATP as a substrate. DOI: http://dx.doi.org/10.7554/eLife.12621.017

    Article Snippet: Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer.

    Techniques: Isolation, Binding Assay, In Vitro, Autoradiography, Staining, SDS Page

    Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro. (A) Schema of the experimental design. ATP hydrolysis-deficient BiP T229A protein was AMPylated in presence of radioactive α- 32 P-ATP with catalytically active GST-FICD E234G coupled to GSH-Sepharose beads. The enzyme-containing beads were removed by centrifugation and excess of non-radioactive ATP was added to the supernatant to competitively displace non-covalently bound α- 32 P-ATP from BiP T229A . Unbound nucleotides were then removed by passing the sample through a desalting column. Trace amounts (1.5 µg) of the recovered 32 P-labeled AMPylated BiP T229A protein were added to excess of unmodified BiP T229A protein (60 µg; a mass ratio of 40:1) and the combined sample was supplemented with ATP and treated for 30 min with increasing concentrations of the SubA protease (0.08 to 120 ng/µl) before denaturing SDS-PAGE, Coomassie staining and autoradiography. ( B ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples described above. In the bottom panel, the Coomassie stain and radioactive signals of the full-length BiP and the substrate binding domain (SBD) were quantified and normalized to the values in lane 1, which were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain signal reports of the fate of unmodified BiP T229A , whereas the radioactive signals report exclusively on the modified BiP T229A in the combined sample. NBD denotes the nucleotide binding domain. ( C ) A shorter exposure (1 hr versus 8 hr) of the autoradiograph shown in “B”, above. It reveals the substantial conservation of the radioactive signal emanating from intact BiP across the time course (the green plot in “B” above), which is obscured by changes in the band width introduced by the progressive digestion of the unlabeled (and unmodified) intact BiP in the sample. DOI: http://dx.doi.org/10.7554/eLife.12621.007

    Journal: eLife

    Article Title: AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

    doi: 10.7554/eLife.12621

    Figure Lengend Snippet: Comparison of the differential susceptibility of unmodified and AMPylated BiP to cleavage by SubA in vitro. (A) Schema of the experimental design. ATP hydrolysis-deficient BiP T229A protein was AMPylated in presence of radioactive α- 32 P-ATP with catalytically active GST-FICD E234G coupled to GSH-Sepharose beads. The enzyme-containing beads were removed by centrifugation and excess of non-radioactive ATP was added to the supernatant to competitively displace non-covalently bound α- 32 P-ATP from BiP T229A . Unbound nucleotides were then removed by passing the sample through a desalting column. Trace amounts (1.5 µg) of the recovered 32 P-labeled AMPylated BiP T229A protein were added to excess of unmodified BiP T229A protein (60 µg; a mass ratio of 40:1) and the combined sample was supplemented with ATP and treated for 30 min with increasing concentrations of the SubA protease (0.08 to 120 ng/µl) before denaturing SDS-PAGE, Coomassie staining and autoradiography. ( B ) Autoradiograph and Coomassie stain (CBB) of an SDS-PAGE gel of BiP from samples described above. In the bottom panel, the Coomassie stain and radioactive signals of the full-length BiP and the substrate binding domain (SBD) were quantified and normalized to the values in lane 1, which were set arbitrarily to 1 for full-length BiP and to 0 for the SBD (graph). The Coomassie stain signal reports of the fate of unmodified BiP T229A , whereas the radioactive signals report exclusively on the modified BiP T229A in the combined sample. NBD denotes the nucleotide binding domain. ( C ) A shorter exposure (1 hr versus 8 hr) of the autoradiograph shown in “B”, above. It reveals the substantial conservation of the radioactive signal emanating from intact BiP across the time course (the green plot in “B” above), which is obscured by changes in the band width introduced by the progressive digestion of the unlabeled (and unmodified) intact BiP in the sample. DOI: http://dx.doi.org/10.7554/eLife.12621.007

    Article Snippet: Radioactive in vitro AMPylation was performed by mixing 7.5 µg of purified wildtype or mutant BiP proteins with 10 µM non-radiolabeled ATP and 0.185 MBq α-32 P-ATP (EasyTide; Perkin Elmer, UK) in a total volume of 30 µl in AMPylation buffer.

    Techniques: In Vitro, Centrifugation, Labeling, SDS Page, Staining, Autoradiography, Binding Assay, Modification

    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,

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

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

    doi: 10.1073/pnas.1319353111

    Figure Lengend 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,

    Article Snippet: Briefly, KaiC hexamers (0.5 mg/mL) were reconstituted from KaiC monomers in the presence of 2 mM [α-32 P]ATP (NEG003H, 370 MBq/mL, 111 TBq/mmol; Perkin Elmer) in MD-2 buffer, followed by incubation at 30 °C in the presence or absence of KaiA (0.1 mg/mL) and KaiB (0.1 mg/mL).

    Techniques:

    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.

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

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

    doi: 10.1073/pnas.1319353111

    Figure Lengend 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.

    Article Snippet: Briefly, KaiC hexamers (0.5 mg/mL) were reconstituted from KaiC monomers in the presence of 2 mM [α-32 P]ATP (NEG003H, 370 MBq/mL, 111 TBq/mmol; Perkin Elmer) in MD-2 buffer, followed by incubation at 30 °C in the presence or absence of KaiA (0.1 mg/mL) and KaiB (0.1 mg/mL).

    Techniques: Incubation