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…

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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.…

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

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…

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…

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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; 

Sensitive Detection of Total Protein on RPPA using Fast Green

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

fast_greenReverse phase protein arrays (RPPA) are designed for sensitive quantitation of a target protein in a very small sample of tissue lysate, and multiplex analysis of many samples in one experiment. This technology holds great promise for the identification and analysis of critical biomarkers for disease states in patient samples and for high-throughput drug screening using cultured cell systems. Quantitative assessment of these biomarkers in each cell lysate spot is highly dependent on a robust normalization method across test subjects in a study.  A common approach for performing normalization within and across RPPA arrays is to base the normalization factor on the total cell lysate protein present in each spot.  This can be accomplished by quantitating the arrayed proteins using stains such as colloidal gold or with SYPRO® Ruby and fluorescence detection (Note: this is typically performed using representative arrays separate than those for antibody biomarker detection).  A cost effective, yet sensitive, fluorescent stain method using Fast Green FCF has also been reported for RPPA cell lysate quantitation down to approximately 100 pg protein per spot using near-infrared detection (Loebke, et al., Proteomics 7, 558-584, 2007) where intrinsic fluorescence of nitrocellulose is negligible.

 

Grace Bio-Labs is developing a near-infrared microarray imager (ArrayCAM, 405nm/800nm) designed for optimal detection on porous nitrocellulose film slides, as discussed in our previous blog article. Using the accompanying method and ArrayCAM, we show sensitive Fast Green protein quantitation down to approximately 8 pg protein per spot on ONCYTE®AVID Porous Nitrocellulose film slides (Fig. 1A and 1B, see attachment for method).  As with many protein stains, it should be noted that the degree of staining is dependent on the chemical makeup of individual proteins, as observed in the lower IgG staining compared to Lysozyme and BSA.  The results of this are inconsequential for RPPAs where staining of a wide population of proteins from whole cell/tissue lysates would not likely be influenced by differences in individual protein staining.

 

With ArrayCAM, we also obtained sensitivity down to 8 pg using printed cell lysate arrays (Fig. 2).  RPPA cell lysates are typically arrayed as 2-fold dilutions with the most concentrated sample spots containing approximately 1000 – 2000 pg of total protein (1-2 mg/ml cell lysate protein, printed with 1 nl volume). The 8 pg sensitivity obtained with Fast Green/ArrayCAM is more than sufficient for typical RPPA sample quantitation, and is comparable to the sensitivity obtained using SYPRO Ruby with 532nm/575nm detection on a GenePix 4400 (Molecular Devices) which resulted in higher signal intensities but also significantly higher background.  The sensitivity reported here for both methods was for the lowest observable cell lysate dilution tested.

 

This is one of various methods being developed for sensitive biomarker detection on porous nitrocellulose film slides using ArrayCAM.  In addition to external normalization methods with Fast Green, use of multichannel fluorescence imagers such as ArrayCAM allow for assay normalization using internal reference standards (ex: β-actin) as well as multi-marker detection on the same microarray spot. For more information regarding porous nitrocellulose and the advantages of near-infrared detection, view our recent presentation at the Microarray World Congress 2013 highlighting these technologies.

 

Figure 1. Fast Green Protein Quantification on ONCYTE® Film Slides with near IR detection using ArrayCam.

A.

Fast Green Protein Quantification on ONCYTE® Film Slides with near IR detection using ArrayCam.

B.

RFY (signal-background vs. (fast green) Protein per microarray spot (ng)

 

Figure 2. Quantitation of Cell Lysates on RPPA using Fast Green with ArrayCAM produces results comparable to Sypro Ruby Protein Stain measured on a GenePix scanner (Molecular Devices, Sunnyvale, CA).

Quantitation of Cell Lysates on RPPA using Fast Green

 

Figure Legends:

Figure 1.  Total protein may be quantified after spotting on ONCYTE® AVID Film Slides by staining with Fast Green and measuring fluorescence using an ArrayCAM, an imaging system developed at Grace Bio-Labs.   ArrayCAM is a CCD-based imaging system using laser excitation at 405 nm. Fast Green emission is 800 nm. (NOTE: Similar results were obtained with ONCYTE® SuperNOVA Film Slides from Grace Bio-Labs.)

A.  Shown is a range of standard proteins from 8 – 500 pg after on-slide staining with Fast Green.

B.  Detection and quantitation of BSA, Lysozyme, and IgG using a Fast Green staining protocol (see attachment) with ArrayCAM are linear down to the lowest protein deposition (8 pg).

 

Figure 2. Fluorescent signal for total protein detection with Fast Green/ArrayCAM yields linear results at cell lysate concentrations commonly used for RPPA analysis down to approximately 8 pg protein per spot.   Similar results were obtained using a standard protein stain method (SYPRO® Ruby total protein stain; 532nm/575nm detection) with the use of a focused-laser microarray scanner (Molecular Devices GenePix 4400).  Cell lysates were from Calyculin A-treated Jurkat cells spotted in 2-fold serial dilution starting with a concentration of 1 mg/ml in a Tris/SDS/Glycerol lysis buffer.

 

See the Lab Protocols Here