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…

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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.…

APPLICATIONS:

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.…

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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,…

APPLICATIONS:

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.

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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…

APPLICATIONS:

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; 

Multiplex Microarrays: The Next Generation ELISA

IN ArrayCAM, Featured Research, ONCYTE nitrocellulose film slides, ONCYTE NOVA, Product Applications, Uncategorized

next-gen-elisa1The hallmark of chronic diseases is the production of highly specific autoantibodies (1,2).  These autoantibodies are increasingly measured to guide clinical decision making.    The goal of these measurements is to define intracellular signal pathway activation profiles. These profiles can be integrated with clinical information to generate “biosignatures” for individual patients, the goal of which is personalized or precision medicine.

 

Comprehensive autoantibody profiling would revolutionize clinical practice at several levels.  Enzyme-linked immunosorbent assays (ELISA) are extensively used in clinical diagnostics for detection of individual analytes in biological samples and are accepted as the “gold standard” against which other assays are measured.   Conventional ELISA methods are limited however, as they only detect one target within a single reaction well.   Confirming the presence of multiple biomarkers within a sample by conventional ELISA requires a multitude of independent tests, increasing time, cost and the possibility of error.   A Microarray is essentially a miniaturized form of ELISA , and offers  multiplexing capacity, significantly  reducing procedure time, labor and sample quantity requirements, making microarrays the  desired format for high-throughput data collection for personalized patient profiling.

Grace Bio-Labs ONCYTE porous nitrocellulose (PNC) offers significant advantages for protein binding over other surface chemistries and is recognized as the surface of choice for protein array applications  (3,4,5).   Grace’s ArrayCAM platform; a combination of ONCYTE PNC , and NIR fluorescent imaging  of  extremely bright and highly stable quantum nanocrystals ,  provides sensitivity and specificity  equivalent to ELISA in an automation friendly format for comprehensive,  high-throughput data collection.

We used PNC to demonstrate multiplex detection of two powerful diagnostic markers ( IL-6 and IL-1alpha)  for inflammation and auto-immune diseases such as Lupus and Osteoarthritis and possibly metastatic cancer (6).   Antibodies were spotted on PNC and challenged with a mixture of antigens.  We selected antibody pairs for detection that did not cross-react.  Detection was without signal amplification ( Table 1 ).  Sensitivity and dynamic range of these non-amplified assays was equivalent to TSA-amplified conventional ELISA’s (Table 1, Fig.2). Enzymatic signal amplification may be used to enhance the sensitivity of detection on both assay platforms and is considered the standard method for conventional ELISA.   Signal amplification has several disadvantages which include added cost, increased number of assay steps, greater assay time and the potential reduction in linear dynamic range.   Table 1 compares the use of signal amplification on LOD and dynamic range.

 

Expansion of multiplex assays to detect multiple antigens in single microarray spots (level 3 multiplexing), cannot employ signal amplification.  Thus, the use of high energy fluorescent tags like Q dots offer significant advantages for multiplex microarrays.

Table 1. Limit of detection and assay range for IL-6 using microarrays compared to conventional ELISA. 

Limit of detection and assay range for IL-6 using microarrays compared to conventional ELISA.

 

Figure  1. Microarray Assays for IL-6 and IL-1α

Microarray Assays for IL-6

Microarray Assays for IL-1α

Figure 2. Multiplexed Microarray Assay for IL-6 and IL-1α

Microarray Assays for IL-6 and IL-1α

 

Figure Legends:

Figure 1.  Microarray assay results using ONCYTE® NOVA PNC film slides for detection of either IL-6 or IL-1α from sample buffer spiked with recombinant antigens.  Antibody pairs for the sandwich assays were purchased from R&D Systems (IL-6: Cat# MAB406/BAF406; IL-1α: Cat# MAB400/BAF400).  Detection was performed after secondary labeling using streptavidin-conjugated QDot800 and imaging with the ArrayCAM microarray imaging system (Grace Bio-Labs).  Sensitivity and assay range for both antigens are consistent to those reported for Quantikine ELISA Kits.   Reported specifications for sensitivity and assay range are: 1.8 pg/ml and 7.8 – 500 pg/ml (IL-6, R&D Systems Cat# M6000B) and 2.5 pg/ml and 4.69 – 300 pg/ml (IL-1α, R&D Systems Cat# MLA00).

Figure 2. Multiplexed microarray assay results using ONCYTE® NOVA PNC film slides for simultaneous detection of IL-6 and IL-1α from sample buffer spiked with recombinant antigens.  Please see Fig. 1 legend for antibody and assay details.

 

References:

1.  Werner, S., et. al (2014) Systematic review: Serum autoantibodies in the early detection of gastric cancer.  Int.  J. Cancer   http://onlinelibrary.wiley.com/doi/10.1002/ijc.28807/abstract

2.  Robinson, WH., et. al (2003) Protein arrays for autoantibody profiling and fine-specificity mapping.  Proteomics 3,2007-2084 http://utzlab.stanford.edu/publications/robinson_proteomics_03.pdf

3.  Liotta, LA et al (2003) Protein Microarrays: Meeting the analytical challenges for clinical applications., Cancer Cell: 2003  http://home.ccr.cancer.gov/ncifdaproteomics/pdf/cancer_cell_protein_arrays.pdf

4. Wagner, JP et al (2013)  Receptor Tyrosine Kinases Fall into Distinct Classes Based on Their Inferred Signaling Networks,  Science Signaling  6: 204 ra58 https://wolfyadlinlab.gs.washington.edu/drupal/sites/default/files/Wagner%2C%20Wolf-Yadlin%20et%20al.%20Sci%20Sign%202013.pdf

5. Gujral, TS et al (2013) Profiling Phospho-signaling Networks in Breast Cancer Using Reverse Phase Protein Arrays. Oncogene: July 18; 32(29): 3470-3476 http://pubmedcentralcanada.ca/pmcc/articles/PMC3670968/pdf/nihms443637.pdf

6.  (2014) First Molecular Target for Triple-Negative Breast Cancer Identified. http://www.genengnews.com/gen-news-highlights/first-molecular-target-for-triple-negative-breast-cancer-identified