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.…

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…

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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,…

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 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; 

Utilizing High Protein Binding Capacity of Nitrocellulose to Increase Sensitivity in Tissue Lysate Arrays (RPPA)

IN Featured Research, ONCYTE nitrocellulose film slides, PATH non-porous nitrocellulose, Product Applications, SuperNOVA, Uncategorized

Utilizing front_Reverse phase protein arraysReverse phase protein arrays (RPPA) offer great promise for the identification and use of critical biomarkers for  drug screening and diagnostics. Samples prepared from tissue lysate that are spotted for RPPA can often produce dilute protein concentrations, sometimes  only a few hundred µg/ml. For single spot depositions in an array, this utilizes less than 1% of the available binding capacity for porous nitrocellulose film. The unsurpassed protein binding capacity of porous nitrocellulose (40 µg/cm2) makes it ideally suited for RPPA and detection of low abundance targets in complex protein mixtures,  and far exceeds the capacity (approx. 500x) of other protein microarray surfaces (Figure 1).

In order to detect low abundance proteins found in tissue lysates, many investigators rely on signal amplification methods that incur increased reagent costs and can potentially confound data due to amplification of non-specific signal.   An alternative approach to signal amplification is to take advantage of the high binding capacity of porous nitrocellulose by performing multiple microarray depositions of dilute samples, and effectively increasing the protein concentration per microarray spot.  Coupled with enhanced signal/noise ratios in the red and near IR emission range (due to lower intrinsic fluorescence for nitrocellulose at these wavelengths), this approach can produce very sensitive detection of RPPAs without the use of signal amplification.  To illustrate this point, we deposited increasing numbers of repetitions from dilute cell lysates (500 µg/ml) for the detection of P-EGFR (Y1173). Detection was performed using either red or near-IR fluorescence scanning and without the use signal amplification (Figure 2).

dilution series of purified IgG-TRITC dissolved in a typical Tris/SDS RPPA lysis buffer

Figure 1.  Shown are dilution series of purified IgG-TRITC dissolved in a typical Tris/SDS RPPA lysis buffer deposited on various protein microarray slide types.  Slides include SuperNOVA porous nitrocellulose, PATH non-porous nitrocellulose, three leading hydrogel providers, and three leading silane-based chemistries (aldehyde, epoxy, amine).  Slides were blocked according to the manufacturer’s recommendations and extensively washed prior to detection by fluorescence of the internal TRITC control at 532nm using a GenePix 4000B (Molecular Devices).

RPPA after detection for p-EGFR (Y1173) with IRDye800 (Li-cor)

 GenePix 4400A (Molecular Devices) for detection of Alexa647 and an InnoScanIR (Innopsys) for detection of IRDye800

Signal-to-Noise obtained with either scanner/dye combination

Figure 2.

(A) Representative scan of an RPPA after detection for p-EGFR (Y1173) with IRDye800 (Li-cor) and scanned using an InnoScanIR (Innopsys).  The duplicate lanes marked “NEG” are cell lysates from untreated A431 cells and the two lanes marked “POS” are cell lysates from EGF-treated A431 cells spotted at a 500 µg/ml concentration.  Spotting rows proceed from 1 deposition (top) to 10 depositions (bottom) of 400 pL each using a Scienion S3 non-contact printer.

(B) Signal obtained from replicate arrays (n=3) using a GenePix 4400A (Molecular Devices) for detection of Alexa647 and an InnoScanIR (Innopsys) for detection of IRDye800.

(C) Signal-to-Noise obtained with either scanner/dye combination highlighting the improved sensitivity in the near infrared over visible wavelengths when using porous nitrocellulose.