Protein Microarrays

Overview

Protein microarrays are tools that can be used in many different areas of research, including basic and translational research. Protein arrays can take on many different formats and can be used to do more than simple expression profiling of samples.

Recent publications have demonstrated that protein microarrays can be used to phenotype leukemia cells, identify novel protein-protein interactions, screen entire proteomes for new proteins, and profile hundreds of patient samples simultaneously.

FAST™ Slides are the premier surface for protein microarray applications. FAST Slides are glass slides coated with a proprietary nitrocellulose polymer. The polymer binds proteins in a non-covalent, irreversible manner and can be probed using the same method as in traditional blots.

FAST Slides are suitable for many types of protein microarrays including antibody arrays and microarray Westerns. There are tremendous advantages to using FAST Slides over traditional ELISAs and Westerns including less sample required, better sensitivity, linearity, and quantitation. The major advantage is that hundreds or thousands of antibodies or samples can be screened simultaneously.

FAST Slides are compatible with fluorescent, chemiluminescent or radiographic detection systems.

KeraFAST® offers kits, reagents, and protocols for scientists who wish to develop their own protein arrays, as well as off-the-shelf arrays and a variety of protein array services.

Microspot ELISA and Antibody Arrays

Microspot ELISA and antibody arrays are used for quantitative profiling of protein expression in cell cultures or clinical specimens. Typically these arrays are low-density (9 to 100 elements/array). In these arrays, known antibodies are arrayed and used to capture antigens from unknown samples. To detect antigen that is bound to the array, the antigen either needs to be labeled directly with a fluorophore, or a second binder/antibody can be used. The latter option creates a sandwich assay similar to a traditional ELISA, only in a microspot format. The KeraFAST FAST Quant Cytokine Quantification Kits offer an example of a microspot sandwich assay.

Single-Capture Antibody Arrays

Single-capture antibody arrays consist of multiple, known antibodies arrayed to a solid surface and used to profile the presence of specific antigens from a pooled sample, usually consisting of both a normal and disease-present sample. A single capture antibody array utilizes a direct or hapten labeling system, which does not require a matched antibody. Single-capture antibody arrays offer a qualitative profiling tool to detect binding events. The KeraFAST Serum Biomarker Chip offers an example of a single-capture antibody array.

Reverse Arrays

Reverse arrays are used to profile dozens or hundreds of samples (research or clinical) for the presence of a small number of antigens (1 to 3). Cell lysates, material from laser capture microdissection or serum samples are arrayed. This creates an array of ‘unknowns’ that can be probed with a small number of antibodies.

Visualization can be performed with a detection or ‘top’ antibody linked to a fluorophore or color detection reagent.

FAST® Pre-Arrayed Assay Kits

• FAST Serum Biomarker Chip Kit
  • Labeling and Detection Systems for Serum Biomarker Chip

• FAST Quant Kits for Cytokine Quantitation

FAST® Protein Array Buffers and Reagents

• Protein arraying buffer
• Protein array blocking buffer
• Protein array wash buffer

FAST® Slide Accessories

• FAST Slide Incubation Chambers
• Slide holders/Slide Frame
• Chip Clip™
• MicroCaster™

FAST® Slide Custom Protein Microarrays

* FAST®, Chip Clip™ and MicroCaster™ are trademarks of the Whatman and GE Healthcare.
**KeraFAST is the exclusive distributor of the FAST Slide related products.

FAST Slide Reference Publications v6

FAST Serum Biomarker Chip Kit

The Serum Biomarker Chip kit enables proteomics researchers to profile and pattern the molecular signature of human serum. The chip addresses the need for a high-throughput technology to enable research in the fields of risk stratification, disease prognosis, drug eligibility, prediction of safety and efficacy, and therapeutic monitoring.

The Serum Biomarker Chip is a single-capture antibody array built on the FAST Slide dual pad platform. Each slide has two identical arrays of antibodies printed in triplicate. Two-color fluorescent detection permits the researcher to reproducibly pattern the relative abundance of 120 human serum proteins between two samples, such as serum samples from diseased and healthy individuals.

Fluorescent labeling is done with the ULS Biotin Single Color Labeling System, available separately. Two-Color Labeling is also available for customers who prefer to do hapten and label swapping experiments. The chip can be scanned with any standard fluorescence scanner. A reusable Slide Holder is also needed, either FAST Frame for 4 slides or Chip Clip for 1 slide.

The Serum Biomarker Chip Kit includes two arrayed 2 pad FAST Slides, two dual Incubation Chambers, 5 ml KeraFAST Protein Array Blocking Buffer, and 125 ml KeraFAST Protein Array Wash Buffer.

We offer two protein fluorescent labeling systems:

• ULS Biotin Single Color Labeling System-- for easy, efficient labeling of proteins, especially for single-capture antibody arrays

The KeraFAST Single Color ULS™ Biotin Labeling Kit is designed to label serum or cell lysate proteins in order to detect their binding to complementary antibodies. The kit is especially intended for use with 2 pad FAST Slides, including the Serum Biomarker Chip and other single-capture antibody chips. The kit contains Universal Linkage System (ULS) chemistry from KREATECH® Biotechnology to label samples containing approximately 100 to 120 µg of protein in serum or plasma or 60 µg protein in a whole cell lysate.

Features

• Highly efficient and uniform labeling of complex protein samples
• Reproducible labeling and signal detection
• Stable, robust, and fast non enzymatic process
• Broad pH range of labeling
• Compatible with most buffers, salts, detergents, and reducing agents
• Labels multiple amino acids

ULS chemistry tags protein methionine, cysteine and histidine residues with biotin (normally at least two biotins per molecule). The attached biotin is then detected with fluorescent streptavidin.

Two-Color Labeling and Detection System - for dual-label hapten swapping applications

The KeraFAST Two-Color Labeling and Fluorescent Detection Kit is designed to label two protein samples. The labeled proteins are pooled and probed against arrayed antibodies in a competitive binding assay and detected using indirect fluorescence.
The kit is intended for use with the 2 pad FAST Slides, including the Serum Biomarker Chip. The kit contains the Universal Linkage System (ULS) chemistry to label samples containing approximately 250 µg of protein in serum, plasma or a whole cell lysate. The kit is designed to label two different protein samples, each with a different hapten. Sufficient labeling reagent is provided to perform a hapten swapping experiment.

Features

• Highly efficient and uniform labeling of complex serum samples
• Reproducible labeling and signal detection
• Stable, robust, and fast non enzymatic procedure
• Reduces pH dependency of labeling efficiency
• System solution includes labeling reagents, fluorescent conjugate, and bench-friendly protocol
• Accounts for hapten-specific differences in either Biotin-ULS or Fluorescein-ULS labeling efficiencies
• Averages differences in antibody-antigen binding interactions caused by steric hindrance
• Minimizes chip-to-chip variability – includes an internal control within the assay

The first pad on the slide is probed with a mixture of two different protein samples, each labeled with a different hapten; the second pad is probed with the same two protein samples but with the haptens reversed. The normalized intensity for each element of each pad is calculated as the average of the biotin and fluorescein labeled derived intensities from a two pad experiment. The ratio between the signal intensity at each spot corresponds to the concentration ratio of the proteins found in the two samples. This method is attractive for antibody chips as it takes into account any hapten-specific differences in antigen-antibody interactions.
The use of the ULS labeling system minimizes background by using indirect fluorescence detection, labels multiple amino acids, and requires no additional materials or reagents.

Each kit includes 20 μL Biotin-ULS, 20 μl Fluorescein-ULS, 80 μl 10x Protein Labeling Buffer, 80 μl 10x KREAstop Solution, 150 μl Streptavidin-DY 647 Conjugate, 350 μl Anti-Fluorescein Antibody-DY 547 Conjugate, 8 Micro Bio-Spin Chromatography Columns, and a User's Manual.

Serum Biomarker Chip Datasheet

FAST Quant kits

Cytokines play an important role in the understanding and treatment of diseases in many medical specialties. Protein microarray technology is accelerating the rate at which researchers can obtain cytokine expression information. Utilizing FAST® Slides by KeraFAST, which are the world’s premier protein arraying surface, quantitative MicroSpot ELISA kits offer researchers standardized solutions with unsurpassed technical expertise and support.

The simple, rapid assays offered by KeraFAST that utilize ELISA sandwich formats to measure concentrations of multiple cytokines from a single biological sample give researchers powerful, versatile and comprehensive solutions.

FAST Quant microspot ELISA for high throughput multiplex cytokine quantification, allows researchers to accurately quantify the concentration of several cytokines in dozens of biological samples simultaneously.

MicroSpot ELISA for High-Throughput Multiplex Cytokine Quantification

FAST Quant™ represents a quantum leap forward in protein microarray technology. With FAST Quant, a researcher can accurately determine the concentration of several cytokines in dozens of biological samples simultaneously, using familiar ELISA immunochemistry.

Built on FAST technology, the high protein binding capacity surface chemistry, FAST Quant combines the power of array technology with the quantitative nature and high-throughput capabilities of traditional ELISA. FAST Quant exhibits greater sensitivity and reproducibility than traditional ELISA. Each FAST Quant kit contains 64 arrays of 8 to 10 monoclonal antibodies with affinities for common human or mouse cytokines. The antibodies are arrayed in a quantitative fashion in triplicate on each array.

Using four 16 pad FAST Slides placed in a FAST Frame (sold separately) FAST Quant offers an 8 x 12 cm footprint – the same as the traditional microplate format. A standard curve can be generated by creating a dilutional series from recombinant antigen mass standards. Due to the solid-phase nature of a microspot assay, it is not necessary to take duplicate measurements of each sample. The MicroSpot is entirely concentration dependent, unlike an ELISA where the reaction is both concentration and volume dependent.
FAST Quant antibody arrays offer a variety of human and mouse menus. Each kit includes four 16 pad FAST Slides prearrayed with one of the cytokine antibody panels, four 16 well incubation chambers, recombinant antigen standard cocktail for standard curve, biotinylated detection antibody cocktail, Whatman Protein Array Blocking Buffer, and Wash Buffer.

A reusable Slide Holder is also needed, either FAST Frame for 4 slides or Chip Clip for 1 slide.

Data analysis is seamless using data reduction software, MicroVigene™ for FAST Quant, the cutting edge utility for protein array image analysis. The software provides rapid data acquisition and comprehensive reports. It analyzes any .tif image from virtually all commercial imaging instruments. The application provides standard curve data, concentrations of unknowns, and percent coefficient of variation for each analyte. FAST Quant is another clear example of Whatman commitment to providing the scientific community with the best solution for multiplex cytokine measurements.

The FAST Frame Slide Holder #10486001 is available separately.
Please see the main FAST Quant page for other kits in this series.

Ordering Information

Cytokines by FAST Quant

FAST® Protein Array Buffers and Reagents

KeraFAST protein array reagents have been optimized for use on FAST Slides. These reagents include Protein Arraying Buffer, Protein Array Blocking Buffer, and Protein Array Wash Buffer.

Protein arraying buffer

• Enhances long-term protein stability and molecular recognition activity
• Enhances fluorescent signal from arrayed sample
• Optimized for use on FAST Slides

Protein array blocking buffer

• Demonstrates highly efficient blocking of protein microarrays
• Exhibits strong reduction of nonspecific antibody-antibody interactions
• Exhibits minimal effects on specific antibody-antigen interactions
• Results in superior signal-to-noise ratios in protein microarray applications
• Superior blocking capabilities

Reduction of nonspecific protein-protein interaction:
Monoclonal capture antibodies were arrayed on FAST Slides and probed with an antibody cocktail consisting of 16 biotinylated polyclonal antibodies followed by Streptavidin-Cy5 detection. Images were taken at an identical laser/PMT setting. The first row of the array field is a positive detection control.

Blocking with Protein Array Blocking Buffer:
Reduction of non specific antibody-antibody interactions by the protein array blocking buffer.

Protein array wash buffer

• Excellent washing buffer for protein microarrays
• Conveniently supplied as 10x concentrate
• Optimized for use on FAST Slide

The Protein Array Wash Buffer has been optimized for efficient washing of protein microarrays to ensure optimum results. This buffer is used as the washing buffer in FAST PAK, FAST Quant, and the Serum Biomarker Chip kits.

FAST® Slide Accessories

Optimal use of FAST Slides is supported by Incubation Chambers, Slide Holders and Protein Array Buffers and Reagents. The FAST PAK Kit combines these items (except Slide Holders) in a convenient starter format. The MicroCaster™ Arrayer allows manual printing of microarrays for labs making a few slides or just starting out.

• FAST Slide Incubation Chambers

KeraFAST array incubation chambers are designed for protein microarray applications on FAST Slides. The chambers provide a convenient way to carry out binding reactions on protein microarrays.

Used in conjunction with the FAST Frame or Chip Clip slide holder, the incubation chambers have a secure gasket design forming a tight, leak-proof seal with the FAST Slides. Simply remove the incubation chamber when the reaction is finished. These reusable chambers are recommended for use at room temperature and elevated incubation temperatures up to 76ºC. Disposable Chamber Covers are also available to prevent splashing and evaporation during incubations.

• Slide Holders

FAST Frame

The FAST Frame multi-slide plate is designed to hold four 16-pad FAST Slides and the corresponding multiwell incubation chambers in a microplate footprint for high-throughput processing of microarrays. The footprint dimensions meet the standards recommended by the Society for Biomolecular Screening.

The 96 well spacing (9 mm center to center) of the array pads on 16-pad FAST Slides makes the loaded FAST Frame compatible with automated liquid handling systems and 8-channel manual pipettors.

Each plate processes up to 64 arrays simultaneously. The rows and columns on each plate are labeled for easy indexing and sample application.

The FAST Frame multi-slide plate is constructed of autoclavable plastic and is compatible with standard 1 x 3" (25 x 76 mm) glass slides when used with KeraFAST reusable incubation chambers (1, 2, 16). The FAST Frame is available as a stand-alone, reusable unit or as a starter kit. The starter kit contains ten 16-pad FAST Slides, ten 16-pad incubation chambers, and 40 chamber covers.

Chip Clip

The Single Grip Slide Holder
The Chip Clip™ securely holds one FAST Slide and incubation chamber for processing multiple arrays simultaneously (includes the Serum Biomarker Chip). Used in conjunction with Whatman incubation chambers, the Chip Clip ensures leak-proof barriers around the arrayed pads on the slide.

The slide and incubation chamber are easily inserted into and removed from the Chip Clip slide holder; side rails hold the chamber firmly against the slide surface. The Chip Clip is compatible with 1 x 3" (25 x 76 mm) slides when used with incubation chambers.

• MicroCaster Arrayer

The MicroCaster™ is an economical, entry-level manual microarraying system for principle and pilot studies. With the MicroCaster 8-pin hand tool, samples can be loaded from 96 well or 384 well microtiter plates.

The MicroCaster slide holder accommodates two slides. It has a built-in indexing system that enables precise printing of up to 768 spots in an array of 32 x 24 spots. It is very easy to set up and use with processing time of 5 to 20 minutes per slide.

The MicroCaster is designed for 1-pad FAST Slides with 20 x 51 mm pad size and is compatible with other slide surfaces.

MicroCaster accessories can be used to increase the flexibility of the manual arrayer system by providing accurate source-plate indexing and reliable pin-tool cleaning.

The MicroCaster accessories are compatible with standard 96 well microplates and they reduce hassle with pin-tool cleaning.

FAST Custom Protein Microarray

The custom array service at KeraFAST enables scientists who have access to microarray scanning and data analysis instrumentation to have FAST Slides printed with proteins from the Whatman antibody menu or from their own protein library.

The custom service provides the scientist complete control of proof-of-principle, assay design, processing, and data analysis of the printed array.

Customer-supplied biological samples are screened against the selected specificities to determine the quantitative or qualitative measurements, depending upon the customer’s requirements. The deliverables to the researcher include access to raw and trimmed data, for each analyte, total fluorescent signal per microspot (quantitative assays only), % CV and standard deviation, and graphed histograms of ratios, signal-background averages, and outlier recognition.

Protein array development projects are driven by certified R&D project leaders responsible for the scope, resources, and timeline that meet the needs of the researcher. Project implementation, project milestones, and customer communications are managed by dedicated account specialists, in collaboration with the research staff.

Protein specificities, whether they are from KeraFAST or customer supplied, are arrayed to the selected FAST Slide configuration – using a quill-pin contact or noncontact printing technology – in triplicate, unless otherwise specified – suspended in KeraFAST protein arraying buffer to achieve long-term stability.

Ordering information:
Item # 100100100