Structured Review

GE Healthcare phyb pcm
Harnessing the At PIF variants for the light-dependent regulation of gene expression in mammalian cells. a The At <t>PhyB</t> <t>PCM</t> and At PIF variants are connected to a VP16 trans -activating domain and an E-protein DNA-binding domain that binds to a synthetic promoter sequence. Red light promotes association of the At PhyB: At PIF pair and thereby activates the expression of a secreted alkaline phosphatase (SEAP) reporter gene. b SEAP expression was determined in Chinese hamster ovary cells (CHO-K1) for the diverse At PIF6 variants and normalized to the constitutive expression of Gaussia luciferase. Black and red bars denote mean ± SEM normalized SEAP expression for n = 4 independent biological replicates under dark or red-light conditions, respectively. Cells were kept in darkness for 24 h, supplemented with PCB, and then either kept in darkness for 24 h or illuminated for 24 h with 20 µmol m −2 s −1 660-nm light. As a negative control, the reporter construct alone was transfected. The numbers above the bars indicate the factor difference between dark and red-light conditions for a given At PIF6 variant. c As b but for the At PIF3 variants. d As b but for the At PIF1 variants.
Phyb Pcm, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 3255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 3255 article reviews
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phyb pcm - by Bioz Stars, 2020-09
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1) Product Images from "Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors"

Article Title: Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors

Journal: Communications Biology

doi: 10.1038/s42003-019-0687-9

Harnessing the At PIF variants for the light-dependent regulation of gene expression in mammalian cells. a The At PhyB PCM and At PIF variants are connected to a VP16 trans -activating domain and an E-protein DNA-binding domain that binds to a synthetic promoter sequence. Red light promotes association of the At PhyB: At PIF pair and thereby activates the expression of a secreted alkaline phosphatase (SEAP) reporter gene. b SEAP expression was determined in Chinese hamster ovary cells (CHO-K1) for the diverse At PIF6 variants and normalized to the constitutive expression of Gaussia luciferase. Black and red bars denote mean ± SEM normalized SEAP expression for n = 4 independent biological replicates under dark or red-light conditions, respectively. Cells were kept in darkness for 24 h, supplemented with PCB, and then either kept in darkness for 24 h or illuminated for 24 h with 20 µmol m −2 s −1 660-nm light. As a negative control, the reporter construct alone was transfected. The numbers above the bars indicate the factor difference between dark and red-light conditions for a given At PIF6 variant. c As b but for the At PIF3 variants. d As b but for the At PIF1 variants.
Figure Legend Snippet: Harnessing the At PIF variants for the light-dependent regulation of gene expression in mammalian cells. a The At PhyB PCM and At PIF variants are connected to a VP16 trans -activating domain and an E-protein DNA-binding domain that binds to a synthetic promoter sequence. Red light promotes association of the At PhyB: At PIF pair and thereby activates the expression of a secreted alkaline phosphatase (SEAP) reporter gene. b SEAP expression was determined in Chinese hamster ovary cells (CHO-K1) for the diverse At PIF6 variants and normalized to the constitutive expression of Gaussia luciferase. Black and red bars denote mean ± SEM normalized SEAP expression for n = 4 independent biological replicates under dark or red-light conditions, respectively. Cells were kept in darkness for 24 h, supplemented with PCB, and then either kept in darkness for 24 h or illuminated for 24 h with 20 µmol m −2 s −1 660-nm light. As a negative control, the reporter construct alone was transfected. The numbers above the bars indicate the factor difference between dark and red-light conditions for a given At PIF6 variant. c As b but for the At PIF3 variants. d As b but for the At PIF1 variants.

Techniques Used: Expressing, Binding Assay, Sequencing, Luciferase, Negative Control, Construct, Transfection, Variant Assay

Architecture and function of plant phytochromes (Phy) and their cognate phytochrome-interacting factors (PIFs). a Modular composition of plant phytochromes. An N-terminal extension (NTE) is succeeded by the photosensory core module (PCM) consisting of consecutive PAS, GAF, and PHY domains, with a phytochromobilin (PΦB) chromophore covalently bound as a thioether within the GAF domain. The C-terminal output module (OPM) comprises two additional PAS domains (PAS-A and PAS-B), succeeded by a histidine-kinase-related domain (HKRD). b In the dark-adapted Pr (red-absorbing) state of the Phy, the PΦB chromophore adopts its 15 Z form. Red light drives isomerization to the 15 E form to give rise to the Pfr state (far-red-absorbing). Vice versa, far-red light drives the Pfr → Pr transition. c In their Pr state (red), plant Phys show no or at most weak interactions with PIFs. Following red-light absorption, the Pfr state (brown) is populated and affinity for the PIFs enhanced. d Modular composition of PIFs. An N-terminal region of around 100 residues contains the so-called APB motif that mediates interactions with phytochrome B. The APB motif further subdivides into the ABP.A and APB.B segments 24 . Certain PIFs also possess a more C-terminal APA motif engaged in interactions with phytochrome A. The C-terminal part comprises a basic helix–loop–helix (bHLH) DNA-binding domain. e Based on the N-terminal fragments of Arabidopsis thaliana PIFs 3 and 6, a panel of PIF variants were generated and probed for light-dependent protein:protein interactions with the PCM of A. thaliana PhyB (cf. Supplementary Table 1 for a detailed description of these derivatives).
Figure Legend Snippet: Architecture and function of plant phytochromes (Phy) and their cognate phytochrome-interacting factors (PIFs). a Modular composition of plant phytochromes. An N-terminal extension (NTE) is succeeded by the photosensory core module (PCM) consisting of consecutive PAS, GAF, and PHY domains, with a phytochromobilin (PΦB) chromophore covalently bound as a thioether within the GAF domain. The C-terminal output module (OPM) comprises two additional PAS domains (PAS-A and PAS-B), succeeded by a histidine-kinase-related domain (HKRD). b In the dark-adapted Pr (red-absorbing) state of the Phy, the PΦB chromophore adopts its 15 Z form. Red light drives isomerization to the 15 E form to give rise to the Pfr state (far-red-absorbing). Vice versa, far-red light drives the Pfr → Pr transition. c In their Pr state (red), plant Phys show no or at most weak interactions with PIFs. Following red-light absorption, the Pfr state (brown) is populated and affinity for the PIFs enhanced. d Modular composition of PIFs. An N-terminal region of around 100 residues contains the so-called APB motif that mediates interactions with phytochrome B. The APB motif further subdivides into the ABP.A and APB.B segments 24 . Certain PIFs also possess a more C-terminal APA motif engaged in interactions with phytochrome A. The C-terminal part comprises a basic helix–loop–helix (bHLH) DNA-binding domain. e Based on the N-terminal fragments of Arabidopsis thaliana PIFs 3 and 6, a panel of PIF variants were generated and probed for light-dependent protein:protein interactions with the PCM of A. thaliana PhyB (cf. Supplementary Table 1 for a detailed description of these derivatives).

Techniques Used: Binding Assay, Generated

Screening At PIF variants for protein:protein interactions with the At PhyB PCM. a The light-adapted Pfr state (brown) of At PhyB thermally recovers to the dark-adapted Pr state (red) in a moderately paced reaction. When binding to an At PIF variant, the recovery reaction is delayed. b At PIF variants were C-terminally tagged with EYFP, expressed in Escherichia coli , cells were lysed, and At PhyB PCM was added to the crude lysate. Samples were exposed to red light, and the recovery reaction was monitored over time by absorption measurements. c Normalized absorption of the At PhyB PCM measured at 720 nm after red-light absorption in the presence of P3.100 (red) or the EYFP-negative control (gray). d The initial rates of the recovery reaction were determined and normalized to the reading obtained for the EYFP-negative control. Data indicate mean ± SEM of n = 3 independent biological replicates.
Figure Legend Snippet: Screening At PIF variants for protein:protein interactions with the At PhyB PCM. a The light-adapted Pfr state (brown) of At PhyB thermally recovers to the dark-adapted Pr state (red) in a moderately paced reaction. When binding to an At PIF variant, the recovery reaction is delayed. b At PIF variants were C-terminally tagged with EYFP, expressed in Escherichia coli , cells were lysed, and At PhyB PCM was added to the crude lysate. Samples were exposed to red light, and the recovery reaction was monitored over time by absorption measurements. c Normalized absorption of the At PhyB PCM measured at 720 nm after red-light absorption in the presence of P3.100 (red) or the EYFP-negative control (gray). d The initial rates of the recovery reaction were determined and normalized to the reading obtained for the EYFP-negative control. Data indicate mean ± SEM of n = 3 independent biological replicates.

Techniques Used: Binding Assay, Variant Assay, Negative Control

Quantitative analyses of the light-dependent protein:protein interaction between At PIF variants and the At PhyB PCM. a In its Pr state, the At PhyB PCM exhibits weak or no affinity to At PIF, but upon red-light exposure, the affinity is enhanced. Binding to the AtPhyB PCM increases the effective hydrodynamic radius of the At PIF variants and slows down rotational diffusion. In turn, the fluorescence anisotropy of an EYFP tag C-terminally appended to the At PIF increases. b Titration of 20 nM P6.100-EYFP with increasing concentrations of dark-adapted (gray) or red-light-exposed At PhyB PCM (red), as monitored by anisotropy of the EYFP fluorescence. Data points show mean of n = 3 biological replicates. The red line denotes a fit to a single-site-binding isotherm. c Absorption spectra of the At PhyB PCM in its dark-adapted Pr state (red line) and as a Pfr/Pr mixture following red-light exposure (blue). The dashed line denotes the absorption spectrum of the pure Pfr state, calculated according to ref. 42 . d As in b but for P3.100-EYFP rather than P6.100-EYFP. Experiments were repeated twice with similar results.
Figure Legend Snippet: Quantitative analyses of the light-dependent protein:protein interaction between At PIF variants and the At PhyB PCM. a In its Pr state, the At PhyB PCM exhibits weak or no affinity to At PIF, but upon red-light exposure, the affinity is enhanced. Binding to the AtPhyB PCM increases the effective hydrodynamic radius of the At PIF variants and slows down rotational diffusion. In turn, the fluorescence anisotropy of an EYFP tag C-terminally appended to the At PIF increases. b Titration of 20 nM P6.100-EYFP with increasing concentrations of dark-adapted (gray) or red-light-exposed At PhyB PCM (red), as monitored by anisotropy of the EYFP fluorescence. Data points show mean of n = 3 biological replicates. The red line denotes a fit to a single-site-binding isotherm. c Absorption spectra of the At PhyB PCM in its dark-adapted Pr state (red line) and as a Pfr/Pr mixture following red-light exposure (blue). The dashed line denotes the absorption spectrum of the pure Pfr state, calculated according to ref. 42 . d As in b but for P3.100-EYFP rather than P6.100-EYFP. Experiments were repeated twice with similar results.

Techniques Used: Binding Assay, Diffusion-based Assay, Fluorescence, Titration

Oligomeric state of the At PIF variants and light-dependent interactions with the At PhyB PCM. a 50 µM At PhyB PCM were exposed to red light and analyzed by size-exclusion chromatography (SEC), where the yellow and red lines represent absorption at 513 and 650 nm, respectively. b As in a but the At PhyB PCM was exposed to far-red light prior to chromatography. c 10 µM P3.100-EYFP were analyzed by SEC. Elution profiles were independent of illumination. d 10 µM of the negative control EYFP were analyzed by SEC. Elution profiles were independent of light. e A mixture of 10 µM P3.100-EYFP and 50 µM At PhyB PCM was exposed to red light and analyzed by SEC. f As in e but samples were illuminated with far-red light, rather than red light. Experiments were repeated twice with similar results.
Figure Legend Snippet: Oligomeric state of the At PIF variants and light-dependent interactions with the At PhyB PCM. a 50 µM At PhyB PCM were exposed to red light and analyzed by size-exclusion chromatography (SEC), where the yellow and red lines represent absorption at 513 and 650 nm, respectively. b As in a but the At PhyB PCM was exposed to far-red light prior to chromatography. c 10 µM P3.100-EYFP were analyzed by SEC. Elution profiles were independent of illumination. d 10 µM of the negative control EYFP were analyzed by SEC. Elution profiles were independent of light. e A mixture of 10 µM P3.100-EYFP and 50 µM At PhyB PCM was exposed to red light and analyzed by SEC. f As in e but samples were illuminated with far-red light, rather than red light. Experiments were repeated twice with similar results.

Techniques Used: Size-exclusion Chromatography, Chromatography, Negative Control

2) Product Images from "The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1"

Article Title: The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1

Journal: Biochemistry

doi: 10.1021/bi101749s

The four Leu region is critical for oligomerization of DCAF1 A. Sequence alignment of putative LisH domains in orthologous DCAF1s. Identical amino acid residues are underlined and the LisH motif, L-X 2 -L-X 3–5 -L-X 3–5 -L is displayed at the bottom. X represents any amino acid. B. FLAG-DCAF1-V5 (encoding residues of 96–1507) was transiently co-expressed with the N-terminally deleted DCAF1 (ΔN-DCAF1-V5, residues of 817–1507), site-directed mutants ΔN-DCAF1 (1LL/EE)-V5, and ΔN-DCAF1 (2LL/EE)-V5, respectively. 1LL/EE corresponds to the Leu850Glu, Leu851Glu double mutant, and 2LL/EE to the Leu852Glu, Leu853Glu double mutant. Cell lysates were immunoprecipitated with anti-FLAG agarose affinity gel. Proteins bound to antibodies were separated by SDS-PAGE, and subjected to immunoblotting with anti-V5 antibody after transfer to nitrocellulose. C. The DCAF1 region corresponding to residues 809–876 and the 1LL/EE and 2LL/EE mutants thereof were expressed and purified from E. coli as thioredoxin (Trx) fusion proteins. Trx and the three Trx-DCAF1 proteins were separated by SDS-PAGE and stained with Coomassie Brillant Blue. D. Multi-angle light scattering of purified Trx-DCAF1 fusion proteins and Trx as a control. Each protein (ca. 2 mg/mL) was injected into an analytical Superdex200 gel filtration column at a flow rate of 0.5 mL/min. The UV (A 280 ) elution profiles of Trx-DCAF1 809–876 WT (▼), Trx-DCAF1 809–876 1LL/EE (●), Trx-DCAF1 809–876 2LL/EE (◆), and Trx (■) are shown. The estimated molecular masses from the scattering data are shown across the elution peaks.
Figure Legend Snippet: The four Leu region is critical for oligomerization of DCAF1 A. Sequence alignment of putative LisH domains in orthologous DCAF1s. Identical amino acid residues are underlined and the LisH motif, L-X 2 -L-X 3–5 -L-X 3–5 -L is displayed at the bottom. X represents any amino acid. B. FLAG-DCAF1-V5 (encoding residues of 96–1507) was transiently co-expressed with the N-terminally deleted DCAF1 (ΔN-DCAF1-V5, residues of 817–1507), site-directed mutants ΔN-DCAF1 (1LL/EE)-V5, and ΔN-DCAF1 (2LL/EE)-V5, respectively. 1LL/EE corresponds to the Leu850Glu, Leu851Glu double mutant, and 2LL/EE to the Leu852Glu, Leu853Glu double mutant. Cell lysates were immunoprecipitated with anti-FLAG agarose affinity gel. Proteins bound to antibodies were separated by SDS-PAGE, and subjected to immunoblotting with anti-V5 antibody after transfer to nitrocellulose. C. The DCAF1 region corresponding to residues 809–876 and the 1LL/EE and 2LL/EE mutants thereof were expressed and purified from E. coli as thioredoxin (Trx) fusion proteins. Trx and the three Trx-DCAF1 proteins were separated by SDS-PAGE and stained with Coomassie Brillant Blue. D. Multi-angle light scattering of purified Trx-DCAF1 fusion proteins and Trx as a control. Each protein (ca. 2 mg/mL) was injected into an analytical Superdex200 gel filtration column at a flow rate of 0.5 mL/min. The UV (A 280 ) elution profiles of Trx-DCAF1 809–876 WT (▼), Trx-DCAF1 809–876 1LL/EE (●), Trx-DCAF1 809–876 2LL/EE (◆), and Trx (■) are shown. The estimated molecular masses from the scattering data are shown across the elution peaks.

Techniques Used: Sequencing, Mutagenesis, Immunoprecipitation, SDS Page, Purification, Staining, Injection, Filtration, Flow Cytometry

Related Articles

Flow Cytometry:

Article Title: Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone
Article Snippet: .. For mass-spectrum analysis, sequential immunoprecipitation and gel filtration, the bound proteins were eluted by incubating with HA peptide (Sigma-Aldrich) at 37°C for 15 min. For gel filtration, the complex was resolved on a Superdex-200 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml min−1 and fractions were collected on a volume base (0.5 ml per tube) throughout the chromatogram. .. Each fraction was concentrated and visualized by SDS–polyacrylamide gel electrophoresis, followed by immunoblotting.

Filtration:

Article Title: Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone
Article Snippet: .. For mass-spectrum analysis, sequential immunoprecipitation and gel filtration, the bound proteins were eluted by incubating with HA peptide (Sigma-Aldrich) at 37°C for 15 min. For gel filtration, the complex was resolved on a Superdex-200 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml min−1 and fractions were collected on a volume base (0.5 ml per tube) throughout the chromatogram. .. Each fraction was concentrated and visualized by SDS–polyacrylamide gel electrophoresis, followed by immunoblotting.

Article Title: Preparation of Recombinant Viral Glycoproteins for Novel and Therapeutic Antibody Discovery
Article Snippet: .. Preparative column: HiLoad 16/60 Superdex 200 prep grade XK 16 gel filtration column (GE Healthcare). .. Analytic column: Superdex 200 10/300 GL gel filtration column (GE Healthcare).

Protease Inhibitor:

Article Title: Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis
Article Snippet: .. Reagents Pfx polymerase was obtained from Invitrogen (Thermo Fisher Scientific, USA); Ni-NTA agarose resin, GAPDH antibodies and RPMI-1640 medium from Sigma ALDRICH (Inc. Sigma-Aldrich Corp, MO, USA); F-12 K medium from ATCC (ATTC, VA, USA); Applixchange-G25M from AppliChem (AppliChem GmbH, Darmstadt, Germany); Superdex-200 from Amersham (GE Healthcare Europe GmbH, Austria); anti-Bid antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); cytochrome c antibodies and Annexin V-FITC Apoptosis Detection kit I from Becton and Dickinson Bioscience (Becton, Dickinson and Company, New Jersey, USA); Protease Inhibitor Cocktail from Promega (Promega Corporation, USA). .. Plasmid construction and mutagenesis cDNA corresponding to human BID (BID(L), isoform 1, 195 aa) was amplified by PCR method.

Size-exclusion Chromatography:

Article Title: Molecular flexibility of DNA as a key determinant of RAD51 recruitment
Article Snippet: .. Multi‐angle light scattering To confirm the monomeric status of F86E RAD51, the peak size‐exclusion chromatography F86E RAD51 elution fraction was serially diluted (5, 1:1 serial dilutions) and loaded onto a Superdex200 10/300 GL size‐exclusion column (GE Healthcare) equilibrated with MALS Buffer (150 mM KCl, 50 mM Tris pH 7.5, 1 mM EDTA, 2 mM DTT). .. Each elution was analysed using a Wyatt Heleos8 + 8‐angle light scatterer linked to a Shimadzu HPLC system comprising LC‐20AD pump, SIL‐20A Autosampler and SPD20A UV/Vis detector.

Purification:

Article Title: A monomeric myosin VI with a large working stroke
Article Snippet: .. A measure of 150–250 μl samples of purified protein at 0.3–0.8 mg ml−1 were applied to a Superdex 200 (30 × 1.5 cm) analytical column (Amersham Pharmacia Biotech) equilibrated in 25 mM NaCl, 10 mM Tris (pH 7.5), 10 mM imidazole (pH 7.4), 2 mM MgCl2 and 1 mM EGTA and controlled using an AKTA purifier (Amersham Pharmacia Biotech). ..

Immunoprecipitation:

Article Title: Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone
Article Snippet: .. For mass-spectrum analysis, sequential immunoprecipitation and gel filtration, the bound proteins were eluted by incubating with HA peptide (Sigma-Aldrich) at 37°C for 15 min. For gel filtration, the complex was resolved on a Superdex-200 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml min−1 and fractions were collected on a volume base (0.5 ml per tube) throughout the chromatogram. .. Each fraction was concentrated and visualized by SDS–polyacrylamide gel electrophoresis, followed by immunoblotting.

Incubation:

Article Title: A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)-SRP Receptor Interaction by the Chloroplast SRP Pathway
Article Snippet: .. The mixture was incubated on ice for specified periods before being loaded onto Superdex 200. ..

other:

Article Title: Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs
Article Snippet: After assembly, Env-NDs were passed through a Yarra 4000, Superdex 200, or Superose 6 10/300 GL column (GE Life Sciences) on an Akta purifier, depending on scale, at room temperature.

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