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; 

Immunoassays: Protein Arrays vs. ELISA and Westerns

IN Discussion Topics, ONCYTE nitrocellulose film slides, Product Applications, Reagents, Uncategorized

Studies of biological systems have expanded beyond the ‘one gene, one protein’ paradigm to the field of proteomics, or studying large numbers of proteins that act in a concert of complex biological systems. Thus in the post-genome era, technology has been driven to deliver multiplex assays that allow us to monitor multiple proteins in parallel, resulting in more biologically relevant information. Multiplex assays are used for basic research and drug discovery to look at thousands of proteins, or a ‘proteome’ in a sample. For clinical diagnostics, monitoring only a few proteins along with controls is sufficient. In both cases, however, specificity, sensitivity, sample availability, reagent costs, and convenience of use are major criteria for selecting a protocol.

Immunoassays are still the preferred platform for most protein studies, particularly clinical diagnostics and drug development,  where specificity is critical . Antibody-based assays including immune-capture in lateral flow devices, immunoblot (Western blot) and ELISA (Enzyme-Linked Immunosorbent Assay) have been around for decades, and are widely adapted into clinical diagnostics.  Most often the first choice is convenience- using the technology that is at hand, and that which has already been set up in the lab or neighboring community. This article describes the relative features and benefits of antibody-based assays, comparing Western, ELISA and protein microarrays.

Western Blots are typically done to determine the presence and integrity of a specific protein. In this assay, a mixture of proteins is first separated based on molecular weight and/or charge by electrophoresis in a gel matrix, then transferred to a membrane and probed with antibody specific to the protein of interest. The relative amount of protein in different samples may be compared and approximated. Because the proteins in a mixture are first separated according to physical properties, the specificity of the Western can be very high, and any cross-reactivity of the detecting antibody with other proteins in the mixture can be distinguished by the known molecular weight of the protein of interest. Western blots are limited to detection of denatured protein because all proteins in the sample are denatured prior to the electrophoresis step. On the downside, Western blots are relatively technically complex, requiring many steps and relatively large sample volume and thus are not easily automated and are relatively time consuming and expensive.

ELISA assays are often performed in 96-well plates and are adaptable to higher throughput than Western blots, but like Western blots offer only monoplex data, or results of a single protein per assay.  Unlike the Western assay, an ELISA can be used to detect native proteins, and protein interactions that require intact three-dimensional structure.  ELISA assays can be highly quantitative when run with a standard curve of the known protein, and well-characterized antibodies.  However, these assays are not highly specific and can give false positives due to cross-reactivity of the detecting antibody with other proteins in the sample. ELISA assays are very useful for looking at protein interactions that require native conformation, and for studying binding competition. In addition, ELISA assays are technically less difficult than Western blots, and can be adapted to higher throughput with automated plate handling and detection systems.

Protein microarrays are more similar to ELISA than Western blot because generally the proteins in a sample are not fractionated prior to the assay.  In constructing microarrays, the proteins are deposited in small (100-300 um) spots on a specially coated microscope slide. The slide is often coated with a polymer like nitrocellulose, or gel that increases the binding capacity of the protein. Microarrays offer advantages of higher throughput, multiplex analysis, low reagent consumption, high sensitivity and lower sample requirement compared to either the Western or ELISA assays (see Tables I and II).

Commercially available protein arrays are now available that offer pre-printed arrays of hundreds to thousands of proteins, typically antibodies used to capture proteins much like an ELISA.  The manufacture of microarrays is in fact a time-consuming and costly process that is not generally feasible for the individual researcher. More importantly, the antibody content spotted onto microarrays is the most valuable and costly component. However, the amount of  antibody required for a microarray as well as the sample requirement is much less than for ELISA. The protein microarray conserves reagents as well as precious samples. Thus for high-throughput, or highly repeated assays the reduced reagent costs, time-savings and sample conservation for microarray can outweigh the relative expense of setup (i.e. printing or purchasing the arrays, see Table II). In addition, the amount of protein or antibody may be limited in some cases (for example, limited amounts of polyclonal antibody or patient sample for diagnostics).

Grace Bio-Labs has developed leading technology for sensitive and high-quality protein microarrays. Our nitrocellulose film-slides provide the highest protein binding capacity,  leading to  excellent sensitivity (down to XX pg/ml). We have developed a suite of reagents designed to optimize results with these arrays in terms of sensitivity (signal/noise) and reproducibility. If you are interested in developing immunoassays in a protein microarray format, we invite you to partner with us with the content of your choice.

Table I: Relative properties of Western Blot, ELISA and Microarray Assays.

Pros

Cons

Best Applications

Western Blot

High specificity

Labor Intensive/time consuming

Denatured protein

Medium quantitation

Low-medium throughput

High sample use

Confirmation of other screening method

Protein-protein interactions

Protein degradation or turnover

ELISA

Medium specificity

High sensitivity

High quantitation

Low variability

Automation potential

Good reproducibility

False positives

High sample use

High reagent use

Time consuming

Labor intensive

Costly setup for automation or high throughput

High throughput screening, automation

Receptor inhibition

Relative quantitation

Ample sample availability

Microarray

Medium specificity

High sensitivity

Highest throughput

Low reagent use

Low variability

Low sample use

Good reproducibility

Multiplex capability

Costly setup for low throughput, mainly costs of scanner and high density arrays.

False positive/negative

High throughput screening

Limited sample availability

Relative quantitation

Multiplex analysis

Proteome assessment

Screening antibody cross-reactivity§

Pathology/diagnosticsŦ

 § Ma et al, 2012 Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology. BMC Biotechnology 2012, 12:88

 Ŧ Duburcq et al., 2004, Peptide−Protein Microarrays for the Simultaneous Detection of Pathogen Infections, Bioconjugate Chem., 2004, 15 (2), pp 307–31

Table II. Costs of Immunoassays/sample

Costs for every immunoassay will depend on the desired sensitivity, the labeling/detection system and particularly the cost of the primary antibody. For assays with abundantly available antibodies, the costs are lower.

Assay

Approximate Costs /sample/analyte

 

Assumptions

Western*

$1.50-10.00

1-2-plex

Depends on digital or film imaging, labeling and detection method.

ELISA**

$5-30

Monoplex

Cost based on efficient use of 96 well plate and commercially available kit.

Microarray

$0.1-30

Multiplex

(up to 100s for discovery, up to 10s for clinical)

Cost based on detection of multiple analytes/slide and use of multiple samples for same assay (same slide content). Depends on labeling and detection (color vs. fluorescence).

 *Li-Cor Corp.

**Udeh et al, 2008 BMC Infectious Diseases 8:174