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); 

CultureWell Removable chamber slide 8 well Catalog #: 103542

Microscopy Reagents

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Grace Bio-Labs microscopy reagents are manufactured in ISO-certified facilities to ensure the highest quality and consistency.

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 8 well Catalog #: 103542

Cell Culture Reagents

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Grace Bio-Labs microscopy reagents are manufactured in ISO-certified facilities to ensure the highest quality and consistency.

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; 

Confocal Fluorescence Microscopy of CRISPR-generated G6PD Knockout mouse liver cells (TIB-73)

IN Cell Culture, Discussion Topics, Featured Research

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas-9 knockout of glucose 6-phosphate dehydrogenase (G6PD)



The first surviving G6PD- knockout somatic cell line was generated with CRISPR-Cas-9 technology to support subcellular localization studies. G6PD,  glucose 6-phosphate dehydrogenase, is the rate-limiting enzyme in the pentose phosphate pathway. G6PD is involved in oxidative stress, apoptosis, angiogenesis and cancer proliferation among other functions (2). In fact, elevated levels of expression and activity of G6PD are frequently observed in breast, colon, endometrial, cervical, prostatic, and lung cancer and G6PD is considered to be a potential therapeutic target. G6PD is also essential for life. Early studies show embryonic lethality of rodent models, indicating that viable G6PD knockout mice cannot be produced.  It was postulated that somatic cells cannot survive knockout of G6PD.  However, this study demonstrates that the mouse hepatic cell line TIB-73 can survive G6PD knockout (1) and that CS16-CultureWell™ Removable Chambered Coverglass (Item # 112358) is the ideally suited to for live-cell imaging experiments in subcellular localization studies.

Cloning and Transfection

TIB-73 cells, an immortalized strongly adherent mouse liver cell line, were grown in 6-well tissue culture plates.  Cloning and transfection of Cas9 and mouse G6PD-targeted sgRNA plasmids (4) were performed as described in Spencer et. al (1). A pTRE2-pur plasmid (3) was co-transfected to confer puromycin resistance to transfected cells.  Puromycin treatment was discontinued 24 hours post-transfection to ensure that the cells would not begin to integrate pTRE2-pur into their genomic DNA.  After puromycin treatment, about 20% of the cells remained alive.  Transfected cells were trypsinized and plated into 96-well plates at a limiting dilution density calculated to be 1 cell per well.  Cells that grew as clonal populations were expanded over time to grow in 6-well plates. These large clonal populations of CRISPR-treated cells were rapidly screened for G6PD activity (5).  G6PD knockout cell populations were identified based on DNA-sequencing and confirmed by Western Blotting.

G6PD subcellular localization studies via live-cell imaging 

Live cell imaging of G6PD knockout cells was utilized to study the subcellular localization of G6PD and to identify the nuclear localization signal. These studies are excellent to study monomeric proteins but for multimeric proteins, the interaction of the native protein with fluorescently tagged-mutants can cause artifacts. Because G6DP is capable of homodimerization G6PD know-out cells were used in this experiment to rule out this interference.  Thus, G6PD knockout cells (Tib73) were propagated on CS16-CultureWell™ Removable Chambered Coverglass (Item # 112358)
and transfected with various plasmid constructs to express full-length G6PD-mCherry, N-terminal-G6PD-mCherry, and C-terminal-G6PD-mCherry mutants. Transfected Tib73 cells were then imaged live by laser scanning confocal microscopy.

Experimental flowchart

Conclusions

The first G6PD-knocked somatic cell line ever produced was used to overcome experimental limitations of subcellular localization studies.  CS16-CultureWell™ Removable Chambered Coverglass is a cost-effective and time-saving solution for this type of experiment. G6PD knockout cells can be cultured directly on the untreated glass bottom of the chamber and transfected with a small volume of reagent.  Fixing and staining, as well as live-cell imaging, can all be performed in the in the same chamber. The G6PD knockout cell line is a convenient tool for studying G6PD role in the survival, proliferation, and metastasis of cancer cells as well as in the development of a potential novel class of inhibitors for cancer therapy.

References

1. Spencer, N. Y., Yan, Z., Cong, L., Zhang, Y., Engelhardt, J. F., and Stanton, R. C. (2016) Definitive localization of intracellular proteins: Novel approach using CRISPR-Cas9 genome editing, with glucose 6-phosphate dehydrogenase as a model, Analytical Biochemistry 494, 55-67.

2. Spencer, N.Y., and Stanton, R.C. (2017) Glucose 6-Phosphate Dehydrogenase and the Kidney, Current Opinion in Nephrology and Hypertension, 26, 43-49.

3. Clontech

4. Lipofectamine LTX with Plus Reagent.

5. Life Technologies Vybrant Cytotoxicity Assay Kit (V-23111).

Cells

G6PD Knockout Cells:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Liver/CRL-3309.aspx

G6PD Wild-type Control (“high-passage”) Cells:

https://www.atcc.org/en/Products/Cells_and_Microorganisms/By_Tissue/Liver/CRL-3308.aspx