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Phosphorimager

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The Phosphorimager is a lab equipment product designed to detect and quantify radioactive signals. It utilizes a phosphor screen to capture and digitize the signal, providing a high-resolution image for analysis.

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Lab products found in correlation

T4 ligase, by New England Biolabs (2 mentions) Emsa binding buffer, by Beyotime (1 mentions) T4 polynucleotide kinase, by New England Biolabs (1 mentions)

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Phosphorimager screen (6 citations)

4 protocols using phosphorimager

1

PABPC1/4 Ubiquitination and RNA Binding

Cited 1 time
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The RNA electrophoresis mobility shift assay (EMSA) was performed as previously described (39 (link)) with some modifications. RNA probe (A)30 was synthesized, and labeled with γ-32P at the 3′-end with T4 Polynucleotide Kinase (NEB, USA) and purified by phenol–chloroform method. Recombinant human PABPC1/4 or ubiquitinated PABPC1/4 (purified from in vivo ubiquitination or in vitro ubiquitination using anti-Flag beads, and eluted with Flag peptide) was incubated with labeled probes in 5× EMSA Binding Buffer (Beyotime, China) at room temperature for 1h, and then the assay was performed on 8% polyacrylamide 0.5× TBE gels to resolve the RNA–protein complexes from the free probes. Gels were then dried and exposed to a phosphor-imager (Kodak, USA) before visualization on a FLA 9000 Fuji scanner (Fujifilm Life Science, USA).
Li C., Han T., Li Q., Zhang M., Guo R., Yang Y., Lu W., Li Z., Peng C., Wu P., Tian X., Wang Q., Wang Y., Zhou V., Han Z., Li H., Wang F, & Hu R. (2021). MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty. Nucleic Acids Research, 49(7), 3796-3813.
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2

p53 Binding Assay with Hemin

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EMSA was performed as described before by Jayaraman and Prives (1995) (link) with slight modifications. The double-stranded DNA probes contained the consensus p53RE sequence (5′-AGG CAT GTC TAG GCA TGT CT-3′ and 5′-AGA CAT GCC TAG ACA TGC CT-3′). The probes were labeled with 32P at the 3′ end with T4 ligase (New England Biolabs). Recombinant human p53 protein (4 μM) was then incubated with hemin at increasing concentrations on ice for 30 min to allow for protein-hemin complex formation. After 30 min incubation on ice, the reaction mixtures were subjected to 6% PAGE analysis, resolving the DNA-protein complexes from the free probes. Gels were then dried and exposed to a phosphorimager (Kodak) before scanning on a FLA 9000 Fuji scanner.
Shen J., Sheng X., Chang Z., Wu Q., Wang S., Xuan Z., Li D., Wu Y., Shang Y., Kong X., Yu L., Li L., Ruan K., Hu H., Huang Y., Hui L., Xie D., Wang F, & Hu R. (2014). Iron Metabolism Regulates p53 Signaling through Direct Heme-p53 Interaction and Modulation of p53 Localization, Stability, and Function. Cell reports, 7(1), 180-193.
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3

p53 Binding Assay with Hemin

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EMSA was performed as described before by Jayaraman and Prives (1995) (link) with slight modifications. The double-stranded DNA probes contained the consensus p53RE sequence (5′-AGG CAT GTC TAG GCA TGT CT-3′ and 5′-AGA CAT GCC TAG ACA TGC CT-3′). The probes were labeled with 32P at the 3′ end with T4 ligase (New England Biolabs). Recombinant human p53 protein (4 μM) was then incubated with hemin at increasing concentrations on ice for 30 min to allow for protein-hemin complex formation. After 30 min incubation on ice, the reaction mixtures were subjected to 6% PAGE analysis, resolving the DNA-protein complexes from the free probes. Gels were then dried and exposed to a phosphorimager (Kodak) before scanning on a FLA 9000 Fuji scanner.
Shen J., Sheng X., Chang Z., Wu Q., Wang S., Xuan Z., Li D., Wu Y., Shang Y., Kong X., Yu L., Li L., Ruan K., Hu H., Huang Y., Hui L., Xie D., Wang F, & Hu R. (2014). Iron Metabolism Regulates p53 Signaling through Direct Heme-p53 Interaction and Modulation of p53 Localization, Stability, and Function. Cell reports, 7(1), 180-193.
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4

Measuring ATPase Activity of hTRiC

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ATPase rates were determined as described in. (Reissmann et al., 2007 (link)). Briefly, ATP hydrolysis by 0.25 μM hTRiC or 0.25 μM hTRiC + 2.5 μM hPFD was measured at 37 °C in ATPase buffer, in the presence of 188 to 1500 M [α−32P]ATP. After 5 min of preincubation, the reaction was started by mixing 6 l [α−32P]ATP solution with 34 l 1.176-fold concentrated reaction mix. At the indicated time points, 2- l samples were taken and transferred onto PEI-cellulose F thin-layer chromatography plastic sheets (EMD Chemicals). The plates were developed in a solvent system containing 1 M LiCl and 0.5 M formic acid in H2O, air-dried and exposed to a phosphorimager (Kodak). After the screen was scanned in a Typhoon 9410 imager, the amount of [α−32P]ATP was quantified using ImageQuant 5.2, amount of [α−32P]/ATP was determined from a standard curve from a dilution set on the thin- layer chromatography sheet.
Gestaut D., Roh S.H., Ma B., Pintilie G., Joachimiak L.A., Leitner A., Walzthoeni T., Aebersold R., Chiu W, & Frydman J. (2019). The chaperonin TRiC/CCT associates with Prefoldin through a conserved electrostatic interface essential for cellular proteostasis. Cell, 177(3), 751-765.e15.
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