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…

APPLICATIONS:

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…

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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…

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…

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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…

APPLICATIONS:

Lorem Ipsum ;  Lorem Ipsum;  Lorem Ipsum; 

Review of Mujawar et al Analytical Chemistry – Distribution of Biomolecules in Porous Nitrocellulose Films

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

An interesting paper in (Analytical Chemistry) describes the use of confocal fluorescence section imaging to analyze protein distribution in microarray spots on nitrocellulose film slides. By scanning and imaging through the Z dimension of a printed spot of fluor labeled protein, a 3-dimensional quantitation of fluorophore density in the films was obtained, showing that commercial nitrocellulose slides from different sources have very different behaviors towards materials applied to their surfaces.

In the study, 3 commercially sourced Nitrocellulose microarray slides were tested: GE Whatman FAST, Sartorius-Stedim Biotech Unisart, and Grace Bio-Labs ONCYTE AVID. Of the 3 types tested, 2 (FAST and Unisart) were measured to have a distribution of biomolecules that demonstrated higher protein density in the outer region of absorption, while one type (ONCYTE AVID) showed a more homogenous distribution throughout the measurement area, suggesting that the interaction of liquid with the membrane is highly dependent on the formulation and chemistry of the nitrocellulose membrane (Figure 1).

CLSM imagesFigure 1. (A) CLSM images of IgG-FITC spots on various NC membrane slides. (B) “Z” stack data for distribution of IgG-FITC spot down into various NC membrane slides. (C) CLSM images of BSA-Alexa spots on various NC membrane slides. (D) “Z” stack data for distribution of BSAAlexa spot down into various NC membrane slides (reprinted with permission from Ref 1. Copyright 2013 American Chemical Society).

Since nitrocellulose slides are marketed as having permeable 3-D matrices providing a scaffold for printed biomolecules to adhere, one might expect that proteins arrayed in a small spot would “soak in” and distribute homogenously through the matrix, but that is apparently not the case. Rather, different film slides show different distributions throughout the “cylinder of analysis”, suggesting that the nitrocellulose formulation drives the way the sample is absorbed into the matrix. The end result can have important effects on the actual assay being performed, as homogenous distribution would be expected to provide more consistent (lower variability) signals than an un-evenly distributed capture analyte.

Figure_2_change in contact angle

Figure 2. Change in contact angle monitored by a “side-view” HSC for a droplet of IgG-FITC in PBS (pH7.4), (reprinted with permission from Ref 1. Copyright 2013 American Chemical Society).

Evidence for the mechanical action of the biomolecule distribution is presented by an analysis of droplet absorption into membranes executed by taking a series of High Speed Camera side view images of liquid drops on membranes in the seconds following deposition (Figure 2). These pictures show very different behaviors by membrane types, with an apparent correlation between maintenance of contact angle during absorption and homogenous distribution through the membrane. This is supported by other studies that show a spreading of droplets in contact with a surface (decreasing contact angle) forces movement of biomolecules in solution towards the air/liquid/surface interface, resulting in heterogeneous biomolecule concentrations in a drying spot, and development of irregular material deposition on the surface (the “doughnut effect”)2,3. In contrast, if a formulation of a surface membrane is such that spots don’t spread (ie contact angle remains high) biomolecule concentration in the drying spot remains relatively homogenous, resulting in more even distribution through the membrane. The ONCYTE AVID slides’ contact angle remained comparatively high during the absorption period, consistent with the observation that the protein was more evenly distributed in this formulation.

Another factor at play that is mentioned but not addressed is the effect of print buffer components on spot drying and biomolecule distribution. Various additives to printing solutions, such as detergents, polymers, salts, or neutral proteins can alter the liquid/surface interaction and change the rate at which water evaporates, causing dramatic differences in the way microarray spots deposit content onto a surface or into a nitrocellulose membrane. Further, these components can have a very large effect on the capture efficiency of bound biomolecular ligands. All these factors add up the conclusion that there is no “one size fits all” solution to protein microarray development, and researchers must carefully examine and test all components of their assay systems to determine the optimally  performing methods for their applications.

Additional information on how to select the optimal nitrocellulose substrate for your application can be found in our Protein Microarray Slide Selection guide.  Please feel free to contact a Grace BioLabs technical support representative for any microarray assay optimization or reagent support at any of our contact options.

References

1)    Mujawar, L.H., Maan, A.A., Khan, M.K.I., Norde, W., van Amerongen, A., Anal. Chem. 2013, 85, 3723-3729.

2)    Ressine, A.; Marko-Varga, G.; Laurell, T.; El-Gewely, M. R. In Biotechnology Annual Review; Elsevier: New York, 2007; Vol. 13, pp 149−200.

3)    Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827−829.