polyethylenimine pei  (Millipore)


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    Structured Review

    Millipore polyethylenimine pei
    Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c <t>/PPD/[PEI(0.5%)-GOx]</t> 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: <t>polyethyleneimine;</t> PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.
    Polyethylenimine Pei, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyethylenimine pei/product/Millipore
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    polyethylenimine pei - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors"

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s19020422

    Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.
    Figure Legend Snippet: Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.

    Techniques Used: Evaporation, Concentration Assay

    2) Product Images from "Inhibition of cell proliferation through an ATP-responsive co-delivery system of doxorubicin and Bcl-2 siRNA"

    Article Title: Inhibition of cell proliferation through an ATP-responsive co-delivery system of doxorubicin and Bcl-2 siRNA

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S135086

    Live/Dead assay of PC-3 cells treated with different nanocomplexes for 24 h: ( A ) control; ( B ) DOX-Duplex; ( C ) PEI25K/DOX-Duplex; ( D ) PEI25K/siRNA and ( E ) PEI/DOX-Duplex/siRNA. Notes: The live and dead cells exhibited green and red fluorescence, respectively. Scale bar 200 µm. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.
    Figure Legend Snippet: Live/Dead assay of PC-3 cells treated with different nanocomplexes for 24 h: ( A ) control; ( B ) DOX-Duplex; ( C ) PEI25K/DOX-Duplex; ( D ) PEI25K/siRNA and ( E ) PEI/DOX-Duplex/siRNA. Notes: The live and dead cells exhibited green and red fluorescence, respectively. Scale bar 200 µm. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.

    Techniques Used: Live Dead Assay, Fluorescence

    The cellular uptake of ternary nanocomplex PEI/DOX-Duplex/siRNA for 6 h through fluorescence microscopic analysis. Notes: ( A ) Red: DOX, ( B ) green: FAM-siRNA, ( C ) blue: DAPI for nuclei staining and ( D ) merge. Scale bar 50 µm. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin; FAM, 5-carboxyflu-orescein; DAPI, 4′,6-diamidino-2-phenylindole.
    Figure Legend Snippet: The cellular uptake of ternary nanocomplex PEI/DOX-Duplex/siRNA for 6 h through fluorescence microscopic analysis. Notes: ( A ) Red: DOX, ( B ) green: FAM-siRNA, ( C ) blue: DAPI for nuclei staining and ( D ) merge. Scale bar 50 µm. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin; FAM, 5-carboxyflu-orescein; DAPI, 4′,6-diamidino-2-phenylindole.

    Techniques Used: Fluorescence, Staining

    Particle size ( A ) and zeta potential ( B ) of different nanocomplexes. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin.
    Figure Legend Snippet: Particle size ( A ) and zeta potential ( B ) of different nanocomplexes. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin.

    Techniques Used:

    Relative activity of caspase-3, -8 and -9 in PC-3 cells after the treatment with different nanocomplexes for 24 h. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.
    Figure Legend Snippet: Relative activity of caspase-3, -8 and -9 in PC-3 cells after the treatment with different nanocomplexes for 24 h. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.

    Techniques Used: Activity Assay

    Agarose gel electrophoresis for the condensation ability of PEI25K toward Bcl-2 siRNA and DOX-Duplex ( A ) and the stability of nanocomplex in the presence of 50% FBS ( B ). Notes: ( A ) Lane 1: DOX-Duplex, lane 2: siRNA, lane 3: PEI/DOX-Duplex at a mass ratio of 1.0, lane 4: PEI/siRNA at a mass ratio of 1.0 and lanes 5 and 6: PEI/DOX-Duplex/siRNA at mass ratios of 1:1:1 and 2:1:1. ( B ) siRNA (lanes 1 and 2), DOX-Duplex (lanes 3 and 4), PEI25K/siRNA (mass ratio of 1.0, lanes 5 and 6), PEI25K/DOX-Duplex (mass ratio of 1.0, lanes 7 and 8) and PEI/DOX-Duplex/siRNA (mass ratio of 1:1:1, lanes 9 and 10) in the absence and presence of 50% FBS. The samples were pretreated with 4 mg/mL heparin before the agarose gel electrophoresis except for lanes 5, 7 and 9. Abbreviations: DOX, doxorubicin; FBS, fetal bovine serum; PEI, polyethylenimine.
    Figure Legend Snippet: Agarose gel electrophoresis for the condensation ability of PEI25K toward Bcl-2 siRNA and DOX-Duplex ( A ) and the stability of nanocomplex in the presence of 50% FBS ( B ). Notes: ( A ) Lane 1: DOX-Duplex, lane 2: siRNA, lane 3: PEI/DOX-Duplex at a mass ratio of 1.0, lane 4: PEI/siRNA at a mass ratio of 1.0 and lanes 5 and 6: PEI/DOX-Duplex/siRNA at mass ratios of 1:1:1 and 2:1:1. ( B ) siRNA (lanes 1 and 2), DOX-Duplex (lanes 3 and 4), PEI25K/siRNA (mass ratio of 1.0, lanes 5 and 6), PEI25K/DOX-Duplex (mass ratio of 1.0, lanes 7 and 8) and PEI/DOX-Duplex/siRNA (mass ratio of 1:1:1, lanes 9 and 10) in the absence and presence of 50% FBS. The samples were pretreated with 4 mg/mL heparin before the agarose gel electrophoresis except for lanes 5, 7 and 9. Abbreviations: DOX, doxorubicin; FBS, fetal bovine serum; PEI, polyethylenimine.

    Techniques Used: Agarose Gel Electrophoresis

    Cell viabilities of PC-3 cells treated with different nanocomplexes for 24 h. Note: Data were expressed as mean value ± SD of three experiments. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin.
    Figure Legend Snippet: Cell viabilities of PC-3 cells treated with different nanocomplexes for 24 h. Note: Data were expressed as mean value ± SD of three experiments. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin.

    Techniques Used:

    Expression level of Bcl-2 and procaspase-3 in PC-3 cells after the treatment with different nanocomplexes for 24 h using Western blot: 1, control; 2, Bcl-2 siRNA; 3, DOX-Duplex; 4, PEI25K/DOX-Duplex; 5, PEI25K/siRNA and 6, PEI/DOX-Duplex/siRNA. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.
    Figure Legend Snippet: Expression level of Bcl-2 and procaspase-3 in PC-3 cells after the treatment with different nanocomplexes for 24 h using Western blot: 1, control; 2, Bcl-2 siRNA; 3, DOX-Duplex; 4, PEI25K/DOX-Duplex; 5, PEI25K/siRNA and 6, PEI/DOX-Duplex/siRNA. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.

    Techniques Used: Expressing, Western Blot

    Flow cytometric analysis of the cell apoptosis of PC-3 cells treated with different nanocomplexes for 24 h: ( A ) control, ( B ) PEI25K/siRNA, ( C ) PEI25K/DOX-Duplex and ( D ) PEI/DOX-Duplex/siRNA. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin; PI, propidium iodide; FITC, fluoresceine isothiocyanate.
    Figure Legend Snippet: Flow cytometric analysis of the cell apoptosis of PC-3 cells treated with different nanocomplexes for 24 h: ( A ) control, ( B ) PEI25K/siRNA, ( C ) PEI25K/DOX-Duplex and ( D ) PEI/DOX-Duplex/siRNA. Abbreviations: PEI, polyethylenimine; DOX, doxorubicin; PI, propidium iodide; FITC, fluoresceine isothiocyanate.

    Techniques Used: Flow Cytometry

    Flow cytometric analysis of cell-cycle arrest in PC-3 cells after the treatment with different nanocomplexes for 24 h: ( A ) control, ( B ) siRNA, ( C ) DOX-Duplex, ( D ) PEI25K/siRNA and ( E ) PEI/DOX-Duplex/siRNA. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.
    Figure Legend Snippet: Flow cytometric analysis of cell-cycle arrest in PC-3 cells after the treatment with different nanocomplexes for 24 h: ( A ) control, ( B ) siRNA, ( C ) DOX-Duplex, ( D ) PEI25K/siRNA and ( E ) PEI/DOX-Duplex/siRNA. Abbreviations: DOX, doxorubicin; PEI, polyethylenimine.

    Techniques Used: Flow Cytometry

    3) Product Images from "Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors"

    Article Title: Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s19020422

    Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.
    Figure Legend Snippet: Graphical representation of two main designs of glucose and lactate biosensors used in this study: GB1 (Panel A ): Pt c /PPD/[PEI(0.5%)-GOx] 5 /PU (5%); GB2 (Panel B ): Pt c /PPD/[PE(0.5%)-GOx] 5 /GTA(1%)-BSA(2%); LB1 (Panel C ): Pt c /PPD/[PEI(0.5%)-LOx] 5 /PU (5%); LB2 (Panel D ): Pt c /PPD/[PEI(0.5%)-LOx] 5 / GTA(1%)-BSA(2%); Pt c : Pt cylinder 1 mm long, 125 μm diameter; GOx: glucose oxidase; LOx: lactate oxidase PPD: poly-ortho-phenylenediamine; PEI: polyethyleneimine; PU: polyurethane; GTA: glutaraldehyde; BSA: bovine serum albumin. The subscript “x” represents the number of dip−evaporation deposition stages and in brackets the concentration of the component.

    Techniques Used: Evaporation, Concentration Assay

    4) Product Images from "A comparative study of three ternary complexes prepared in different mixing orders of siRNA/redox-responsive hyperbranched poly (amido amine)/hyaluronic acid"

    Article Title: A comparative study of three ternary complexes prepared in different mixing orders of siRNA/redox-responsive hyperbranched poly (amido amine)/hyaluronic acid

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S32676

    Characterization of the synthesized novel redox-responsive hyperbranched poly(amido amine) (named PCD): ( A ) proton nuclear magnetic resonance spectrum of PCD, ( B ) acid-base titration curves of PCD and polyethylenimine (PEI), ( C ) cytotoxicity of PCD and PEI on MCF-7 human breast cancer cells, and ( D ) cytotoxicity of PCD and PEI on MDA-MB-231 human breast cancer cells.
    Figure Legend Snippet: Characterization of the synthesized novel redox-responsive hyperbranched poly(amido amine) (named PCD): ( A ) proton nuclear magnetic resonance spectrum of PCD, ( B ) acid-base titration curves of PCD and polyethylenimine (PEI), ( C ) cytotoxicity of PCD and PEI on MCF-7 human breast cancer cells, and ( D ) cytotoxicity of PCD and PEI on MDA-MB-231 human breast cancer cells.

    Techniques Used: Synthesized, Nuclear Magnetic Resonance, Titration, Multiple Displacement Amplification

    5) Product Images from "Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor"

    Article Title: Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S144764

    General scheme of the whole-cell bioluminescence biosensor for detection of oligomerization. Note: Schematic representation explains split-luciferase strategy for monitoring of A53T αS oligomerization and using of this system for screening of GQDs effect on A53T αS oligomerization. Abbreviations: αS, alpha-synuclein; PEI, polyethylenimine; GQDs, graphene quantum dots.
    Figure Legend Snippet: General scheme of the whole-cell bioluminescence biosensor for detection of oligomerization. Note: Schematic representation explains split-luciferase strategy for monitoring of A53T αS oligomerization and using of this system for screening of GQDs effect on A53T αS oligomerization. Abbreviations: αS, alpha-synuclein; PEI, polyethylenimine; GQDs, graphene quantum dots.

    Techniques Used: Luciferase

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    Article Title: Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes
    Article Snippet: .. For DNA complex formation, 25 kDa branched polyethylenimine (bPEI; Sigma-Aldrich) was dissolved in reduced serum medium OptiMEM (Fisher Scientific) and then added dropwise to DNA in OptiMEM, vortexed for 10 s, and incubated for 15 min at room temperature. ..

    Molecular Weight:

    Article Title: Visualization of MMP-2 Activity Using Dual-Probe Nanoparticles to Detect Potential Metastatic Cancer Cells
    Article Snippet: .. Chemicals Poly(dl -lactide-co-glycolic acid) (PLGA, lactide/glycolide 50:50, Molecular weight, M w = 38,000–54,000) and polyethylenimine (PEI, branched, M w = ~25,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N -(lissamine rhodamine B sulfonyl) (RhoB lipid) was acquired from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

    Article Title: Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications
    Article Snippet: .. Poly(ethylene glycol) with a molecular weight of 5000 Da (PEG-5K) and branched polyethylenimine with molecular weight of 25,000 Da (PEI-25K) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fluka (Buchs, Switzerland), respectively. .. N ,N ′-azobis (isobutyronitrile) (AIBN, 98%, Shanghai Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) was recrystallized twice in methanol prior to use.

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    Millipore tlc pei cellulose f plates
    Effect of amino acid withdrawal on [ 32 P]guanyl nucleotide content of RagC-containing heterodimers endogenous to 32 P i -labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation ( IP ) was performed using nonimmune ( NI ) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by <t>TLC</t> on <t>PEI</t> cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori , origin.
    Tlc Pei Cellulose F Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tlc pei cellulose f plates/product/Millipore
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    tlc pei cellulose f plates - by Bioz Stars, 2020-08
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    94
    Millipore polyethyleneimine
    Scheme of synthesis of <t>polyethyleneimines</t> (PEI)-n.
    Polyethyleneimine, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyethyleneimine/product/Millipore
    Average 94 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    polyethyleneimine - by Bioz Stars, 2020-08
    94/100 stars
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    Image Search Results


    Effect of amino acid withdrawal on [ 32 P]guanyl nucleotide content of RagC-containing heterodimers endogenous to 32 P i -labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation ( IP ) was performed using nonimmune ( NI ) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by TLC on PEI cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori , origin.

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: Effect of amino acid withdrawal on [ 32 P]guanyl nucleotide content of RagC-containing heterodimers endogenous to 32 P i -labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation ( IP ) was performed using nonimmune ( NI ) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by TLC on PEI cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori , origin.

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Labeling, Incubation, Immunoprecipitation, Thin Layer Chromatography

    Rag heterodimers lack detectable GTPase activity in vitro . A , the RagB/C wild-type heterodimer does not hydrolyze [γ- 32 P]GTP in vitro . GST-Ha-Ras WT (▵), GST-Ha-Ras G12V (▴), and GST-RagB WT /FLAG-RagC WT (●) were charged with [γ- 32 P]GTP and examined for intrinsic GTPase activity. The amount of unhydrolysed [γ- 32 P]GTP on proteins was measured at intervals thereafter by nitrocellulose filter binding assay. B , a recombinant catalytic fragment of NF1 stimulates GTP hydrolysis by Ha-Ras WT but not Ha-Ras G12V or RagB WT /C WT . GST-Ha-Ras WT (●, dashed line ), GST-Ha-Ras G12V (○, dashed line ), or GST-RagB WT /FLAG-RagC WT (●, solid line ) were charged with [γ- 32 P]GTP, and a catalytic fragment of NF1 was added. The release of 32 P i at 30 °C was measured at intervals thereafter by the charcoal method. C , neither RagB wild-type or RagC wild-type within a heterodimer hydrolyze [α- 32 P]GTP detectably in vitro . GST and various GST-RagB/FLAG-RagC heterodimers were charged with [α- 32 P]GTP, and at intervals thereafter, nucleotides were extracted and separated on TLC on PEI cellulose. D , extracts from HEK293T cells do not promote the ability of RagB/C to hydrolyze [γ- 32 P]GTP. HEK293T cells were lysed in the absence of detergent ( solid line ) or with CHAPS (0.3%, dotted line ), TritonX-100 (1%, short dashes ), or RIPA buffer (Pierce) ( long dashes ). GST-Ha-Ras WT ( triangles ) and GST-RagB WT /FLAG-RagC WT ( circles ) charged with [γ- 32 P]GTP were mixed with each of these extracts at 30 °C, and the amount of protein-bound [γ- 32 P]GTP was measured by filtration through nitrocellulose filters. E , MonoQ anion exchange chromatography of a HEK293T cell extract contains GAP activity toward Ha-Ras wild-type, not RagB/C wild-type. HEK293T cells were extracted by freezing and thawing and separated by anion exchange chromatography. Each fraction was incubated at 30 °C with GST-Ha-Ras WT (▵, dashed lines ), GST-Ha-Ras G12V (▴, dashed lines ), and GST-RagB WT /FLAG-RagC WT , each charged with [γ- 32 P]GTP. After 30 min, protein-bound [γ- 32 P]GTP was measured as in C .

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: Rag heterodimers lack detectable GTPase activity in vitro . A , the RagB/C wild-type heterodimer does not hydrolyze [γ- 32 P]GTP in vitro . GST-Ha-Ras WT (▵), GST-Ha-Ras G12V (▴), and GST-RagB WT /FLAG-RagC WT (●) were charged with [γ- 32 P]GTP and examined for intrinsic GTPase activity. The amount of unhydrolysed [γ- 32 P]GTP on proteins was measured at intervals thereafter by nitrocellulose filter binding assay. B , a recombinant catalytic fragment of NF1 stimulates GTP hydrolysis by Ha-Ras WT but not Ha-Ras G12V or RagB WT /C WT . GST-Ha-Ras WT (●, dashed line ), GST-Ha-Ras G12V (○, dashed line ), or GST-RagB WT /FLAG-RagC WT (●, solid line ) were charged with [γ- 32 P]GTP, and a catalytic fragment of NF1 was added. The release of 32 P i at 30 °C was measured at intervals thereafter by the charcoal method. C , neither RagB wild-type or RagC wild-type within a heterodimer hydrolyze [α- 32 P]GTP detectably in vitro . GST and various GST-RagB/FLAG-RagC heterodimers were charged with [α- 32 P]GTP, and at intervals thereafter, nucleotides were extracted and separated on TLC on PEI cellulose. D , extracts from HEK293T cells do not promote the ability of RagB/C to hydrolyze [γ- 32 P]GTP. HEK293T cells were lysed in the absence of detergent ( solid line ) or with CHAPS (0.3%, dotted line ), TritonX-100 (1%, short dashes ), or RIPA buffer (Pierce) ( long dashes ). GST-Ha-Ras WT ( triangles ) and GST-RagB WT /FLAG-RagC WT ( circles ) charged with [γ- 32 P]GTP were mixed with each of these extracts at 30 °C, and the amount of protein-bound [γ- 32 P]GTP was measured by filtration through nitrocellulose filters. E , MonoQ anion exchange chromatography of a HEK293T cell extract contains GAP activity toward Ha-Ras wild-type, not RagB/C wild-type. HEK293T cells were extracted by freezing and thawing and separated by anion exchange chromatography. Each fraction was incubated at 30 °C with GST-Ha-Ras WT (▵, dashed lines ), GST-Ha-Ras G12V (▴, dashed lines ), and GST-RagB WT /FLAG-RagC WT , each charged with [γ- 32 P]GTP. After 30 min, protein-bound [γ- 32 P]GTP was measured as in C .

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Activity Assay, In Vitro, Filter-binding Assay, Recombinant, Thin Layer Chromatography, Filtration, Chromatography, Incubation

    The effect of amino acid withdrawal and insulin on the [ 32 P]guanyl nucleotide content of stably expressed RagC WT and RagC S75L heterodimeric complexes in 32 P i -labeled HeLa cells. A , B , and C , replicate plates of HeLa cells stably expressing GFP-streptag, GFP-streptag-RagC WT , or GFP-streptag-RagC S75L were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml), for another 2 h. In A and B , insulin (1.0 μ m ) was added to some of the cells in DMEM ( AA +/ I ) 30 min before harvest. The nucleotides bound to Strep-Tactin ( strept ) pull-downs were extracted and separated by TLC on PEI cellulose. One set of 32 P -labeled HeLa cells expressing FP-streptag, treated as described above for AA-, AA+, and AA+/I, were rinsed, extracted directly into acetonitrile, and then the solubilized total nucleotides were separated by TLC. The 32 P comigrating with GDP and GTP was quantitated by phosphorimaging. After subtraction of the averaged values found in the GFP-streptag lanes, the percentage of [ 32 P]GTP was calculated as [[ 32 P]GTP / (1.5 × [ 32 P]GDP + [ 32 P]GTP)], and the averaged values are shown. In A , the top and center panels on the right show GFP immunoblot analyses of the cell lysates and representative Strep-Tactin pull-downs (corresponding to lanes 1 , 4 , and 7 of each set). The bottom panel shows an immunoblot analysis of S6K(T389P) corresponding to lanes 1 , 4 , and 7 of each set. In B , the top panel on the right shows a GFP immunoblot analysis of the lysates, whereas the center and bottom panels show lysate immunoblot analyses for 4E-BP(T37P/T46P) and PRAS40(S246P), respectively. In C , one set of 32 P-labeled HeLa cells expressing GFP-streptag, treated as in Fig. 6 , were rinsed, extracted directly into HClO 4 (0.3 m , 0 °C, HClO 4 extract ). The HClO 4 supernatants were neutralized with KHCO 3 , and the [ 32 P]guanyl nucleotides were quantified as above. In addition, the nucleotides in the lysate after pull-down of the Strep-Tactin beads were also analyzed. Immunoblot analyses of the lysates and representative Strep-Tactin pull-downs are shown in the right panel. Ori , origin; std , guanyl nucleotide standards; Ins , insulin; ACN , acetonitrile; Ct , C-terminal.

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: The effect of amino acid withdrawal and insulin on the [ 32 P]guanyl nucleotide content of stably expressed RagC WT and RagC S75L heterodimeric complexes in 32 P i -labeled HeLa cells. A , B , and C , replicate plates of HeLa cells stably expressing GFP-streptag, GFP-streptag-RagC WT , or GFP-streptag-RagC S75L were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml), for another 2 h. In A and B , insulin (1.0 μ m ) was added to some of the cells in DMEM ( AA +/ I ) 30 min before harvest. The nucleotides bound to Strep-Tactin ( strept ) pull-downs were extracted and separated by TLC on PEI cellulose. One set of 32 P -labeled HeLa cells expressing FP-streptag, treated as described above for AA-, AA+, and AA+/I, were rinsed, extracted directly into acetonitrile, and then the solubilized total nucleotides were separated by TLC. The 32 P comigrating with GDP and GTP was quantitated by phosphorimaging. After subtraction of the averaged values found in the GFP-streptag lanes, the percentage of [ 32 P]GTP was calculated as [[ 32 P]GTP / (1.5 × [ 32 P]GDP + [ 32 P]GTP)], and the averaged values are shown. In A , the top and center panels on the right show GFP immunoblot analyses of the cell lysates and representative Strep-Tactin pull-downs (corresponding to lanes 1 , 4 , and 7 of each set). The bottom panel shows an immunoblot analysis of S6K(T389P) corresponding to lanes 1 , 4 , and 7 of each set. In B , the top panel on the right shows a GFP immunoblot analysis of the lysates, whereas the center and bottom panels show lysate immunoblot analyses for 4E-BP(T37P/T46P) and PRAS40(S246P), respectively. In C , one set of 32 P-labeled HeLa cells expressing GFP-streptag, treated as in Fig. 6 , were rinsed, extracted directly into HClO 4 (0.3 m , 0 °C, HClO 4 extract ). The HClO 4 supernatants were neutralized with KHCO 3 , and the [ 32 P]guanyl nucleotides were quantified as above. In addition, the nucleotides in the lysate after pull-down of the Strep-Tactin beads were also analyzed. Immunoblot analyses of the lysates and representative Strep-Tactin pull-downs are shown in the right panel. Ori , origin; std , guanyl nucleotide standards; Ins , insulin; ACN , acetonitrile; Ct , C-terminal.

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Stable Transfection, Labeling, Expressing, Incubation, Thin Layer Chromatography

    Phosphorylation of dThd, N5 ( 3 ), N5-Br ( 12a/b ), N5-I ( 4a/b ), and N5-I 4 ( 5 ) by recombinant TK1. Assay products were separated by PEI–cellulose TLC.

    Journal: Inorganic Chemistry

    Article Title: Synthesis, biological evaluation, and radioiodination of halogenated closo-carboranyl thymidine analogues

    doi: 10.1021/ic202150b

    Figure Lengend Snippet: Phosphorylation of dThd, N5 ( 3 ), N5-Br ( 12a/b ), N5-I ( 4a/b ), and N5-I 4 ( 5 ) by recombinant TK1. Assay products were separated by PEI–cellulose TLC.

    Article Snippet: The reaction mixture was centrifuged and 2 μL sample portions were spotted on PEI–cellulose TLC plates (EMD Chemicals Inc.).

    Techniques: Recombinant, Thin Layer Chromatography

    Phosphorylation rates of dThd, N5-2OH and N3-substituted dThd analogues IPwith hTK1. Assay products were separated by PEI–cellulose TLC and quantified by β -radiography. ( A ). Compounds 3e, 3f and 7e-7g ; (a) dThd monophosphate (dTMP), (b)

    Journal: European journal of medicinal chemistry

    Article Title: Synthesis of N3-Substituted Carboranyl Thymidine Bioconjugates and their Evaluation as Substrates of Recombinant Human Thymidine Kinase 1

    doi: 10.1016/j.ejmech.2012.11.041

    Figure Lengend Snippet: Phosphorylation rates of dThd, N5-2OH and N3-substituted dThd analogues IPwith hTK1. Assay products were separated by PEI–cellulose TLC and quantified by β -radiography. ( A ). Compounds 3e, 3f and 7e-7g ; (a) dThd monophosphate (dTMP), (b)

    Article Snippet: The reaction mixtures were centrifuged and 2 μL sample portions were spotted on PEI–cellulose TLC plates (EMD Chemicals Inc.).

    Techniques: Thin Layer Chromatography

    Scheme of synthesis of polyethyleneimines (PEI)-n.

    Journal: Polymers

    Article Title: Influence of Cross-Linking Degree on Hydrodynamic Behavior and Stimulus-Sensitivity of Derivatives of Branched Polyethyleneimine

    doi: 10.3390/polym12051085

    Figure Lengend Snippet: Scheme of synthesis of polyethyleneimines (PEI)-n.

    Article Snippet: The acetylated derivative of branched polyethyleneimine (PEI-0), namely, poly-N-isobutyroylethyleneimine, was obtained by acylating polyethyleneimine with isobutyroyl chloride under the conditions of the Einhorn reaction (with methylene chloride solvent and triethylamine acceptor).

    Techniques: