Protein Crystallization

Protein crystallization is a key assay for structural studies of proteins. The protocols for crystallization of protein are challenging due to the stringent requirement for pure samples and control of environmental conditions during the crystallization process. Vapor diffusion using hanging drop is a preferred method for obtaining quality crystals with…

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Protein Microarray Substrates

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Grace Bio-Labs microarray surface chemistry is based on the well-known protein-binding properties of nitrocellulose. A range of different formulations have…

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Antigen-Capture AssayAntibody Capture AssayRPPA- Reverse Phase Protein MicroarrayLaser micro-dissection RRPAEpitope-mappingBiomarker Discovery and ValidationImmunogen Discovery

DNA Microarray Substrates

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Epoxy Microarray Slides provide a uniform substrate for a variety of DNA/RNA-based diagnostic applications.

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DNA/Oligonucleotide Microarray ;  microRNA Microarray ;  Single Nucleotide Polymorphism (SNP) Analysis ;  Gene Expression Profiling; 

Microarray Reagents

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Grace Bio-Labs microarray regents have been specifically formulated to achieve the full potential of porous nitrocellulose, accelerating experimental design and…

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Antigen-Capture Assay Antibody Capture Assay RPPA- Reverse Phase Protein Microarray Laser micro-dissection RRPA Epitope-mapping Biomarker Discovery and Validation Immunogen Discovery

APPLICATIONS:

Antibody capture assay ;  Epitope-mapping ;  Biomarker Discovery and Validation ;  Immunogen Discovery ;  Quantitative multiplex immunoassays ;  Peptide Microarrays ;  Autoantibody profiling ;  Multiplex serological assays; 

ProPlate®

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ProPlates® were specifically designed to enable automated robotic liquid handling. Two main configurations are available: The ProPlate® Microtiter Plate is comprised…

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ProteomicsProtein MicroarraysProtein expression analysisAntibody profiling cDNA and oligonucleotide arrays

NanoParticle Fluorescent Calibration Slide

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Photostable nanoparticles arrayed on glass slides for calibration of fluorescence imaging systems and quantitative analysis.

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Calibration of Microarray Scanners ;  Quantitative Microarray Analyses Microscope Focal Plane Adjustment;  Microscope Focal Plane Adjustment; 

CoverWell Perfusion Chambers

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CoverWell ™ perfusion press-to-seal covers form water-tight, multiwell cell incubation or cytochemistry chambers when pressed to coverslips or microscope slides.…

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Live-cell imagingMicroscopyImagingSingle molecule spectroscopy

SecureSeal™ Hybridization Chambers

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SecureSeal™ Hybridization Chambers are thin, silicone-gasketed chambers providing optimal surface-to-volume fluid dynamics for hybridization assays on large or multiple specimens…

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In situ hybridizationProtein and DNA MicroarraysImmunocytochemistryRapid microfluidic prototypingFluorescence Resonance Energy Transfer (FRET)

CoverWell™ Incubation Chambers

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CoverWell™ incubation chambers are reusable, easy to apply chambers that attach without the use of adhesive.  CoverWells™ enclose a large…

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Reverse Transfection Microarray;  DNA Microarray;  In-situ hybridization;  Immunohistochemistry; 

Silicone Isolators

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Silicone Isolators allow researchers to isolate specimens using removable hydrophobic barriers. They may be used to isolate cells grown in…

SecureSeal™ Hybridization Chambers

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SecureSeal™ Hybridization Chambers are thin, silicone-gasketed chambers providing optimal surface-to-volume fluid dynamics for hybridization assays on large or multiple specimens…

HybriWell™ Sealing System

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HybriWell™ Sealing System bonds securely to a microscope slide surface in seconds to confine small reagent volumes with samples and…

Hybridization and Incubation

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Hybridization and incubation Seals ad Chambers from Grace Bio-Labs are ideally suited for in situ-hybridization assays. The adhesive seal of…

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In-situ hybridization MicroarraysFluorescence In situ Hybridization (FISH)FRET (Fluorescence Resonance Energy Transfer)

FastWells™ Reagent Barriers

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FastWells™ are sticky, flexible silicone gaskets that form hydrophobic reagent barriers around specimens without messy adhesives or special slides. Gaskets may…

FlexWell™ Incubation Chambers

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FlexWell™ incubation chamber silicone gaskets form wells on slides using clean release adhesive to isolate up to 16 specimens per…

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Protein MicroarrayHybridizationIncubation

HybriSlip™ Hybridization Covers

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HybriSlips™ are rigid, light-weight, thin plastic coverslips that minimize friction and facilitate uniform reagent distribution during incubation steps which require…

ProPlates®

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ProPlates® were specifically designed to enable automated robotic liquid handling. Two main configurations are available: The ProPlate® Microtiter Plate is comprised…

APPLICATIONS:

ProteomicsProtein MicroarraysProtein expression analysis;  Antibody profiling ;  cDNA and oligonucleotide arrays; 

Silicone Isolators™ Sheet Material

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Silicone isolator™ sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. Where additional sealing is…

APPLICATIONS:

Protein and DNA arrays ;  Immunohistochemistry;  Fluorescence In situ Hybridization (FISH) ;  Biopolymers and hydrogel formulation ;  Cryogenic-transmission electron microscopy (Cryo-TEM) ;  Microwave crystallization ;  Ultra-small-angle X-ray scattering (USAXS) ;  Tissue ingeneering;  Live cell lithography” (LCL); 

Imaging Spacers

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Imaging spacers are ultra-thin adhesive spacers which peel-and-stick to coverglass or microscope slides to confine specimens without compression. Layer multiple…

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Imaging;  Microscopy;  High-temperature single-molecule kinetic analysis;  Anti‐Stokes Raman scattering microscopy; 

CoverWell™ Imaging Chambers

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CoverWell ™ imaging chambers are designed to stabilize and support thick and free-floating specimens for confocal microscopy and imaging applications.…

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Confocal microscopy Imaging Tissue and Cell staining ;  High Resolution Microscopy ;  Live-cell imaging ; 

CoverWell™ Perfusion Chambers

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CoverWell ™ perfusion press-to-seal covers form water-tight, multiwell cell incubation or cytochemistry chambers when pressed to coverslips or microscope slides.…

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Single molecule spectroscopy Live-cell imaging Microscoscopy

FastWells™ Reagent Barriers

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FastWells™ are sticky, flexible silicone gaskets that form hydrophobic reagent barriers around specimens without messy adhesives or special slides. Gaskets may…

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Microscopy Fluorescence In situ Hybridization (FISH) Single-molecule fluorescence analysis ;  Immunohistochemistry ; 

MultiSlip™ Coverglass Inserts

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MutliSlip™ inserts with 8 (18mm x 18mm) or 15 (12mm x 12mm) No. 1.5 German glass coverglass per insert are…

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High resolution microscopy Fluorescent imaging Immunohistochemistry ;  Cell Culture; 

SecureSeal™ Adhesive Sheets

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These adhesive sheets are made using the same SecureSeal™ adhesive as is used to make HybriWell™ and SecureSeal™ Incubation Chambers.  Thin,…

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Imaging ;  Tissue and Cell staining ;  High Resolution Microscopy; 

SecureSlip™ Silicone Supported Coverglass

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SecureSlip™ Silicone Supported Coverglass is affixed to a thin microscopically transparent silicone base which secures it to culture vessels by…

Imaging and Microscopy

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Imaging seals and chambers from Grace Bio-Labs offer a selection of tools for cell/tissue staining for high quality results in…

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Tissue and Cell stainingHigh Resolution MicroscopyLive-cell imaging

CultureWell removable chamber slide

CultureWell™ Removable Chamber Slide

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CultureWell- removable chamber slide allows the cultivation and analysis of cells directly on a borosilicate microscope slide.

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Fluorescence microscopy ;  Confocal microscopy ;  Cell differentiation and transfection;  Immunohistochemistry ;  Immunofluorescence;  Immunostaining;  Tissue and cell staining ;  Fluorescence Resonance Energy Transfer (FRET) Microscopy; 

CultureWell™ MultiWell Chambered Coverslips

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CultureWell™ chambered coverglass products consist of removable and reusable, non-cytotoxic silicone gaskets secured to number 1.5 German coverglass. Chambered coverglass…

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Cell Culture Fluorescence applications In-situ hybridization Immunostaining

CS16-CultureWell™ Removable Chambered Coverglass

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CS16 CultureWell™ removable chambered coverglass is a 16-well chambered coverglass cell culture vessel, with 2 x 8 format with standard…

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Cell CultureFluorescence applicationsIn-situ hybridizationImmunostaining

CultureWell™ Coverglass Inserts

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Each CultureWell™ coverglass insert is comprised of four chambered coverglass, assembled in a disposable frame placed in a standard 86mm…

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High resolution microscopy Fluorescent imaging Immunohistochemistry

CultureWell™ Reusable Gaskets

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Gaskets are ideal for forming wells on glass microscope slides or in polystyrene dishes. Gaskets are non-sterile and may be…

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Cell CultureHigh resolution microscopyFluorescent imaging Immunohistochemistry

CultureWell™ Silicone Sheet Material

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CultureWell™ clear silicone sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. They may be…

APPLICATIONS:

Cell CultureHigh resolution microscopy Fluorescent imagingImmunohistochemistry

MultiSlip™ Coverglass Inserts

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MutliSlip™ inserts with 8 (18mm x 18mm) or 15 (12mm x 12mm) No. 1.5 German glass coverglass per insert are…

APPLICATIONS:

Cell CultureFluorescent imaging Immunohistochemistry

SecureSlip™ Silicone Supported Coverglass

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SecureSlip™ Silicone Supported Coverglass is affixed to a thin microscopically transparent silicone base which secures it to culture vessels by…

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Cell CultureImmunofluorescence assayMicroscopy

CultureWell™ ChamberSLIP 16, Non-Removable Chambered Coverglass

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CultureWell™ NON Removable Chambered Coverglass, 16 Well, No. 1.5 German borosilicate Coverglass. Product consists of cell culture vessels, with a…

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Cell Culture Fluorescence applicationsSmall volume incubation Immunostaining

Silicone Wound Splints

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Wound splints are constructed of silicone and include suture sites for increased precision in affixing on or within an animal…

Silicone Isolator Sheet Material

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Silicone isolator™ sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. Where additional sealing is…

APPLICATIONS:

Protein and DNA arrays ;  Immunohistochemistry ;  Fluorescence In-situ Hybridization (FISH) ;  Biopolymers and hydrogel formulation;  Cryogenic-transmission electron microscopy (Cryo-TEM) ;  X-ray scattering ;  Microwave crystallization ;  Ultra-small-angle X-ray scattering (USAXS) ;  Tissue engineering Live cell lithography (LCL); 

CultureWell Silicone Sheet Material

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CultureWell™ clear silicone sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. They may be…

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Lorem Ipsum ;  Lorem Ipsum;  Lorem Ipsum; 

GENEBLAZER®FRET CELL-BASED ASSAY ON CS-16 REMOVABLE CHAMBERED COVERGLASS

The GeneBLAzer® FRET Cell-based Assay provides a reliable method to study cellular pathways and cellular responses to potential drugs. The technology is based on Fluorescence Resonance Energy Transfer (FRET), a ratiometric detection method that reduces experimental artifacts enhancing assay accuracy.

The regulation and transmission of extracellular chemical signals to the nucleus are vital for DNA transcription and gene expression. The mammalian Janus Kinase signal transducers and activator of transcription (JAK/STAT) is the principal signaling mechanism for a number of cytokines and growth factors. JAK activation stimulates cell proliferation, differentiation, and apoptotic cell death. (1). Disruption of the JAK/ STAT signal transduction pathway has been shown to lead to leukemia, inflammatory disease, and erythrocytosis.

In hematopoietic cells, the JAK2/STAT5 signaling pathway plays an essential role in blood cell formation in response to cytokines such as Granulocyte-macrophage colony-stimulating factor  (GM-CSF), Interleukin-3 (IL-3) and Erythropoietin (EPO)(4). In this pathway, binding of these cytokines to their respective cell surface receptors results in the activation of JAK2, which in turn phospho-activates STAT5 proteins at specific tyrosine residues(3).

TF-1 is a cell line, derived from a patient with erythroleukemia, useful for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors. TF1 cells have an intact GM – CSF – JAK2 – STAT5 pathway (2). The CellSensor® irf1 – bla TF1 Cell Line contains a beta-lactamase reporter gene under control of the irf – 1 response element stably integrated into TF1 cells. CellCensor IrF1-bla TF-1 cell line was used, in this study, to assess beta-lactamase inhibition.

Introduction

Intracellular Beta-Lactamase inhibition was analyzed in CellSensor® IrF1-bla TF-1 cell lines with the Live Blazer- FRET B/G substrate. A membrane permeable beta-lactamase substrate labeled with two different fluorophores was used to detect beta-lactamase activity. The two fluorophores bound to the beta-lactamase substrate, coumarin, and fluorescein, form an efficient FRET pair. In the absence of beta-lactamase activity exciting the coumarin at 409 nm in the intact molecule results in FRET (Fluorescence Resonance Energy Transfer) to the fluorescein, which emits a green fluorescence signal at 520 nm. The presence of beta-lactamase activity, however, results in cleavage of the substrate spatially separating the two dyes and disrupting FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm.

Materials and Methods

Growth media: RPMI 1640 (Gibco),  10% dialyzed FBS (Sigma-Aldrich), 1mM Sodium Pyruvate (Invitrogen), 100 U/mL Penicillin (Sigma-Aldrich), 100 μg/mL Steptomycin (Sigma-Aldrich), 5 μg/mL Blasticidin (Invitrogen), 2ng/mL Recombinant Human GM-CSF (Invitrogen), 0.1 mM non-essential aminoacids (Invitrogen).

Assay media: Opti-MEM (Gibco), 0.5% dialyzed FBS (Sigma-Aldrich), 0.1 mM Non Essential aminoacids (Invitrogen), 1mM Sodium Pyruvate (Invitrogen), 100 U/mL Penicillin (Sigma-Aldrich), 100 μg/mL Steptomycin (Sigma-Aldrich), 2 ng/mL Recombinant Human GM-CSF (Invitrogen),

LiveBLAzer-FRET B/G loading kit (Invitrogen).

Cell Culture

Cells were cultured on a CS16 Removable Chambered CoverGlass (Grace Bio-Labs, item# 112358) at 37°C in RPMI 1640 medium supplemented with 10–20% fetal bovine serum (Invitrogen) and maintained in a humidified atmosphere of 5% CO2.

Irf-bla Cellsensor Assay

Cells were harvested from growth media, centrifuged at 400 g for 5 minutes and resuspended in assay media at a density of about 4×105 cells/mL and incubated at 37°C, 5% CO2 for 16-20 hours. Cells were then resuspended at 1 · 106 cells/ml in assay medium without blasticidin, seeded at 8 · 104 cells/well into a plated in a CS16 CultureWell Removable chambered coverglass and incubated with decreasing concentration of Potassium clavulanate, Sigma-Aldrich Corp. St. Louis, MO, USA,  0, 0.1, 10μM in 0.1% DMSO for 16 h. Following 5 to 6 h of assay incubation, 8 µL/well of 6× LiveBLAzer-FRET B/G (Invitrogen) substrate was applied to each well and incubated for 2 to 3 h at room temperature. Fluorescence was measured at 409 (ex.) nm/518 nm (em).

Results

Visual assessment of the effects of decreasing beta-lactamase inhibitor was performed through fluorescence microscopy. The change in fluorescence due to disruption of FRET (Fluorescence Resonance Energy Transfer) is proportionate to the concentration of added inhibitor as shown in figure 1.

Figure 1 Effect of decreasing concentrations of beta-lactamase inhibitor using CellSensor® Irf1-bla F-1 cell-based assay using LyticBLAzer TM –FRET B/G Reagent (Life TechnologyInc. Carlsbad, CA).

Conclusions

 CS16 Removable chambered Coverglass provide the ideal surface for culturing and imaging sensitive cell lines, including stably transfected cell lines specifically engineered to study pathway specific responses. Beta-lactamase inhibition was accurately and efficiently analyzed thanks to the CS16 Removable chambered Coverglass which proved to be ideally suited for FRET- fluorescence applications since the black gasket at the bottom of the chamber helps to minimize light scatter, enhancing the fluorescence signal.

Acronyms used in the text: 

JAK: Janus Kinase

STAT: Signal Transducer and Activator of Transcription

GM-CSF: Granulocyte-macrophage Colony-Stimulating Factor

IL-3: Interleukin-3

EPO: Erythropoietin

DMSO: Dimethyl sulfoxide

FBS: Fetal Bovine Serum

FRET: Fluorescence Resonance Energy Transfer

References

(1)Jeffrey J. Babon, et al., The molecular regulation of Janus kinase (JAK) activation. Biochem J. 2014 Aug 15; 462(1): 1–13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112375/

(2) Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A, Takaku F. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol. 1989 Aug; 140 (2):323-34. https://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&db=PubMed&list_uids=2663885&dopt=AbstractPlus

3) Gillinder KR, Tuckey H, Bell CC, Magor GW, Huang S, Ilsley MD, Perkins AC. Direct targets of pSTAT5 signaling in erythropoiesis. PLoS One. 2017 Jul 21; 12 (7) https://www.ncbi.nlm.nih.gov/pubmed/28732065

(4) Zhu X1,2, Liu R3,4, Guan J5, Zeng W6, Yin J2, Zhang Y2. Jak2a regulates erythroid and myeloid hematopoiesis during zebrafish embryogenesis. Int. J Med Sci. 2017 Jul 18;14 (8):758-763 https://www.ncbi.nlm.nih.gov/pubmed/28824311