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.

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…

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Lorem Ipsum ;  Lorem Ipsum;  Lorem Ipsum; 

Sensitive and Accurate Multi-Color RPPA Using Grace Bio-Labs ArrayCAM Protein Microarray System

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

Introduction

Profiling sets of multiple protein markers and signaling pathways is becoming the standard method for biomarker discovery, accurate disease diagnosis, and monitoring of therapeutic response. Low-cost, high throughput multiplexed assay platforms that can provide specific, sensitive, and reproducible measurements, featuring reliable and robust assay controls along with easy to use methodologies will yield increased discovery throughput and decreased disease mortality.The application of Reverse Phase Protein Array (RPPA) has developed into a valuable multiplexed tool for the precise study of cell signaling. Detection of low abundance proteins from small or labile samples is a primary obstacle for RPPA users while enzyme catalyzed colorimetric detection is the traditional method for obtaining measurements of sufficient sensitivity for quantitation of these proteins of interest. However, accuracy of robust multi-color inter- and intra- experimental data normalization for consistent measurements is prohibitively challenging with single channel colorimetric detection. Further, dynamic range of colorimetric detection is significantly curtailed. Grace Bio-Labs has developed a complete proteomic microarray system incorporating high protein-binding porous nitrocellulose (PNC), multi-color multiplexing using near IR- nanocrystal detection, optimized reagents and protocols, along with rapid image acquisition and automated software analysis to address the needs of RPPA users for both sensitivity as well as accuracy (Figure 1).

3-channel assay detection

 

Method

 

Platform Description – The Grace Bio-Labs ArrayCAM™ system employs a suite of components specifically optimized to function together for easy, rapid, sensitive and accurate RPPA.  ONCYTE PNC film is employed to immobilize cell lysates, providing 500X greater binding capacity than other microarray substrates (Figure 2).  This translates into maximum signal to noise (S/N) and 50X lower LOD.  Porous nitrocellulose is optimal in combination with near-IR detection modalities due to emission and excitation light resonant scattering.  Optimized pore size and structure amplify emitted fluorescent signal achieving a 150-fold signal enhancement (Figure 3.)  When considering fluorescence detection, the inherent native PNC auto-fluorescence at visible wavelengths must be considered.  Migrating detection to far-red and near infra-red wavelength mitigates PNC auto-fluorescence and provides 4x greater S/N than visible wavelengths (Figure 4).  Optimal fluorescence in the near IR range is provided by quantum nanocrystals QNC which are bright, photo-stable, protein sized fluorescent inorganic particles.  The QNC is excited at a single wavelength, while emitting in narrow emission bands, facilitating robust multi-color multiplexing at red, far red and near-IR wavelengths (Figure 5).  Furthermore, these particles do not quench or photo-bleach under repeated scanning or degrading environmental conditions such as at high ozone levels, providing an archivable record of experimental data.  Finally, to compliment the unique single excitation/multiple emission wavelength properties of (QNC) the low cost multi-wavelength reading ArrayCAM instrument provides array image data in under 1 minute.  Following acquisition the automated software rapidly provides spot finding and data analysis results, reliably eliminating lengthly manual and subjective data processing.  This novel system exploits the properties of PNC and QNC, using the three channels simultaneously to increase throughput and integrate total protein normalizers, improving data accuracy (Figure 6). 

Three Color RPPA – To illustrate the benefits of high throughput RPPA three-color multiplexing arrays, two microarrays containing Raf 1 kinase inhibitor time-course/dose-response treated C32 cell lysate arrays were probed with antibodies for three markers and detected with QNC.  One array was probed with anti-Erk, total and phosphorylated (T202/Y204), and the second with anti-S6, total and phosphorylated (S235/236) then both detected with Q800 and Q655, respectively.  Both arrays were also simultaneously probed for GAPDH for total protein normalization and detected with Q585.  Excellent time-course dosage patterns are observed (Figure 7) with both changing (phosphoprotein) and stable (non-phosphorylated) markers clearly exhibited.  As expected, signal for P-Erk drops rapidly in response to the Raf 1 kinase inhibitor and remains low over time.  The temporal effect on the downstream P-S6 is clearly observed at the later timepoints. Cellular levels of the non-phosphoryated proteins remain constant across dose and time, indicating that the observed changes in phosphorylation states are not due to general decreases in Erk or S6. 

Additionally, to demonstrate the benefit of using intra-spot multi-color signal normalization , data from cell lysate replicate spots (n=8, each cell lysate) for P-pERK were normalized four different ways; 1) Un-normalized, 2) normalized to GAPDH measured on another slide from the same print run, 3) normalized to total protein signals using FastGreen protein stain on another slide from the same print run and 4) normalized to intra-spot GAPDH measured on the same experimental slide using the third (Q585) channel.  For cell lysates on this array, intra-spot normalization provided the best replicate consistency (average CV = 10.1%) when compared to no normalization (average CV = 17.1%), external total protein normalization (average CV = 13.1%) and external GAPDH protein normalization (average CV = 17.1%) (Figure 8).  This illustrates how implementing intra-spot normalization mitigates typical variability inherent with microarray printing and customary assay processing providing the most accurate signal quantitation within and between experiments.

 

Near IR Detection Systems Comparison – Multi-color near-IR detection is not a novel technique with other suppliers offering near-IR organic fluors and confocal microarray scanners capable of performing similar multi-color methods.  Here, we compare a complex existing system to the faster and less expensive ArrayCAM system.  Multi-color detection using quantum nanocrystals (QDots) and LI-COR probes were compared on the same C32 cell lysate time-point arrays as previously described.  Slides were probed with P-ERK, cPARP or P-S6 antibodies (normalized with GAPDH) then detected with multiple wavelength probes for both QDots and LI-COR respectively.  For QDot detection, the slides were imaged in under 1 minute per color using the ArrayCAM.  For LI-COR probes the slides were imaged in 5 minutes per color using an Innopsys 710-IR scanner.  The QDot/ArrayCAM method proved as or more sensitive than the more widely used and expensive LI-COR/Innopsys method, detecting the same signaling changes and in some cases detecting them earlier (Figure 9).  This demonstrates the rapid and lower cost ArrayCAM/QDot method is equivalent and in some cases superior to the LI-COR/Innopsys system in terms of sensitivity and ease of use.

Quantum Nanocrystal versus Amplified Colorimetric Detection – The absolute protein mass detection sensitivity was demonstrated between quantum nanocrystals (QDot) and colorimetric detection using DAB with catalyzed signal amplification.  Both systems were employed to detect GAPDH in arrayed serially titrated NIH 3T3 and Jurkat cell lysates of known total protein concentrations.  The colorimetric system is traditionally performed using an auto-stainer instrument.  Therefore, in the present case, the QDot assay was observed using both manual benchtop and automated staining methods.  Sensitivity was determined for each method using respective signal minus background (S-B) of replicate blank plus two standard deviations to derive an LOD using linear regression against the S-B cell lysate titration series of known concentrations.  Results obtained using unamplified QDots were consistently more sensitive than the standard amplified colorimetric detection system (Figure 10).

Conclusions

 

We have shown that the Grace Bio-Labs ArrayCAM Microarray System provides a valuable sensitive, accurate, easy to use and economical tool for investigators or clinicians in their study of multiple protein signaling pathways or detection and monitoring of important clinical biomarkers.  The system delivers sensitivity equivalent or superior to current benchmark RPPA methods for detection of low-abundance proteins.  Utilizing multi-wavelength detection assay methods provides improved data normalization accuracy and higher throughput not possible using standard methodologies.  This translates into improved consistency and more robust data sets within and between assays.  Rapid, easy to use instrumentation and software present a low barrier to adoption when compared to more complex and costly systems.  This facilitates rapid implementation and greatly accelerates access to data and publication.  

 

Technical Resources

 

Institute Curie Reverse Phase Protein Array Platform –

http://www.institut-curie.org/recherche/reverse-phase-protein-array-platform

 

Reverse-Phase Protein Array (RPPA) Assay System User Guide –

http://www.gracebio.com/media/RPPA_Assay_System_Guide_1.0.pdf

 

Assay Kinetics for Quantum Dots on Protein Arrays –

http://www.gracebio.com/research/assay-kinetics-for-quantum-dots.html

 

Reverse phase protein array based tumor profiling identifies a biomarker signature for risk classification of hormone receptor-positive breast cancer – Sonntag, et al. doi:10.1016/j.trprot.2014.02.001

http://www.sciencedirect.com/science/article/pii/S2212963414000023

 

Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions – Wang et al. Proteome Science 2011, 9:53 – doi:10.1186/1477-5956-9-53

http://www.proteomesci.com/content/9/1/53

 

Multiplex Protein Arrays with Quantum Dots

http://www.gracebio.com/research/multiplex-protein-arrays.html

 

References

 

  1. Espina, Virginia, Lance A. Liotta, and Emanuel F. Petricoin III. “Reverse-phase protein microarrays for theranostics and tatient tailored therapy.” Tumor Biomarker Discovery. Humana Press, 2009. 89-105.
  2. Becker, Karl-Friedrich.  “Using tissue samples for proteomic studies – Critical considerations.” PROTEOMICS-Clinical Applications (2015).
  3. Wang, Xiaoyu, et al. “Improved protein arrays for quantitative systems analysis of dynamic signaling pathway interactions.” Proteome Science 9.1 (2011): 1-13
  4. Calvert, Valeri S., et al.  “Development of multiplexed protein profiling and detection using near infrared detection of reverse-phase protein microarrays.” Clinical Proteomics 1.1 (2004): 81-89
  5. De Luca, Antonella, et al.  “The RAS/RAF/MEK/ERK and the PI3K/AKT signaling pathways: role in cancer pathogenesis and implications for therapeutic approaches.”  Expert opinions on therapeutic targets 16.S2 (2012): S17-S27