Arrayit Protein A/G Microarray Substrate Slides (Box of 25) provide an advanced recombinant protein A/G surface for the microarray market, featuring polished atomically smooth glass surface, corner chamfer for unambiguous side orientation, ground edges, covalently-attached and durable protein A/G surface chemistry for binding human immunoglobulin G (IgG) and other human immunoglobulins, uniformly-spaced protein A/G molecules, high binding capacity and low background fluorescence, standard glass substrate slide format (1 x 25 x 76 mm), box of 25.
Microarray Substrates & Slides - Protein A/G Microarray Substrate Slides for Binding Human Immunoglobulin G (IgG) Molecules
Arrayit Protein A/G Microarray Substrate Slides contain a highly uniform monolayer of Recombinant Protein A/G over the surface of the substrate for microarray applications. The surface is designed to bind human immunoglobulin proteins IgG1, IgG2, IgG3, IgG4 and all types of total IgG. The open platform glass slide format (1 x 25 x 76 mm) makes this product line compatible with all standard microarray printers, scanners, hybridization stations and other slide processing and detection equipment.
Table of Contents
- Introduction
- Quality Control
- Product Description
- Protocol Protein Microarray
- Technical Specifications
- Recommended Equipment
- Technical Assistance
- Troubleshooting Tips
- Scientific Publications
- Ordering Information
- Storage Conditions
- Warranty
Introduction
Congratulations on taking a big step towards improving the affordability, quality and speed of your genomics, biomedical, pharmaceutical and agricultural research. This booklet contains all the information required to take full advantage of Arrayit Protein A/G Microarray Substrate Slides.
Quality Control
Arrayit takes every measure to assure the quality of our Arrayit Super Microarray Substrate Slides. The finest microarray biochip cleanroom research was used to develop these products. Rigorous quality control monitoring on a substrate-by-substrate basis guarantees that each these products conforms to the highest industry standards.
Product Description
This surface starts with ArrayIt SuperClean glass then it is activated with Protein A/G. Gene fusion of the Fc-binding domains of Protein A and Protein G has resulted in production of a structurally and functionally chimeric protein with broader binding than either Protein A or Protein G alone. During fusion, the Protein G gene sequence coding for the serum albumin-binding site is eliminated. This protein is expressed in E. coli and secreted into the surrounding medium during fermentation. The product obtained is consistent in quality and yield because the bacterial host is engineered to be deficient in major proteolytic activities. Binding is less pH-dependent than either Protein A or Protein G alone, occurring well at pH 5-8. The extended Fc-binding properties of Protein A/G make it a popular tool in the investigation and purification of human immunoglobulins. Arrayit Protein A/G Microarray Substrate Slides bind to all human IgG subclasses, IgA, IgE, IgM and to a lesser extent IgD; however, Arrayit Protein A/G does not bind mouse IgA, IgM or murine serum albumin. Arrayit Protein A/G is an excellent tool for purification and detection of mouse monoclonal antibodies from IgG subclasses without interference from these other serum proteins. Individual subclasses of mouse monoclonals are most likely to have stronger affinity to this chimeric protein than to either Protein A or Protein G. The Protein A/G on the substrate slide is covalently bound to the surface for highly durable performance, high signal strength and low background. Arrayit SuperClean Microarray Substrate Slides provide the foundation for this product line, providing a highly polished glass surface with an optical smoothness tolerance of 50 angstroms over the entire 1 x 25 x 76 mm surface area for optimum homogeneity; this new surface sets the highest quality standard for Protein A/G-activated substrate slides for microarray applications. These substrate slides are packed in white slide boxes, vacuum packed and should be stored at 4°C before and after use.
Short Protocol Protein (Steps 1-7)
1. Suspend protein samples in Protein Printing Buffer at 0.25-1 µg/µl.
2. Print protein samples onto Protein A/G Substrates.
3. Block with BlockIt buffer and then wash and dry the printed microarrays.
4. React the processed microarrays with fluorescent samples.
5. Wash the microarrays to remove un-reacted fluorescent material.
6. Scan the microarray to produce a fluorescent image.
7. Quantitate and model the fluorescent data.
Complete Protocol Protein (Steps 1-7)
1. Suspend protein samples to be spotted onto the Protein A/G Microarray Substrate Slide in 2X Protein Printing Buffer for a final protein concentration of 0.25-1.0 µg/µl. Perform transfers carefully to prevent protein denaturation. Mix samples thoroughly prior to printing and transfer into 384-well microplates at 5-10 µl per well. Keep samples cold (4°C) at all times during sample preparation.
2. Print protein samples onto Protein A/G Substrates. Use a Protein Edition Microarrayer such as the SpotBot® Pro or NanoPrint Protein Edition to print the protein samples on the Protein A/G Microarray Substrate Slides. Place the 384-well microplates onto the platen of the instrument and print the samples onto the Protein A/G Microarray Substrate Slides. For best results, make sure the microplates remain at 4°C for the duration of the print run. Following the print run, incubate the printed substrate slides on the robot platen for 1 hour at ambient humidity to allow binding to the slide surface.
3. Activate the printed microarrays by mixing for 60 min in Protein Microarray Activation Buffer (Cat. PMAB). The activation step can be performed under a cover slip or in batch mode using 450 ml of Protein Microarray Activation Buffer with gentle mixing in a High-Throughput Wash Station. Following activation, wash the slides two times for 5 min in Protein Microarray Wash Buffer (Cat. PMWB) and one time for 1 sec in Protein Microarray Rinse Buffer (Cat. PMRB). All washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station (Cat. HTW ) with gentle agitation. After washing, spin for 1 sec in a Microarray High-Speed Centrifuge to dry the slides.
4. React the activated microarrays with sample of choice. Samples can be prepared by mixing 1-5 µg of labeled protein with Protein Microarray Reaction Buffer (Cat. PMRB), making sure not to dilute the Protein Microarray Reaction Buffer by more than 1.5-fold. The same volume of protein sample should be used for all reactions in which comparative information is sought. Reactions can be performed using LifterSlip™ (elevated cover slips) or multi-well reaction cassettes. Binding reactions may also be performed using large sample droplets or a variety of custom gaskets. Undiluted or diluted samples can be used depending on the sample concentration and protein expression level. Typically 5 µg of labeled protein is sufficient to obtain strong signals. Binding reactions should be allowed to proceed for 1-4 hours. Following the reaction step, wash the microarrays three times for 3 min in Protein Microarray Wash Buffer (Cat. PMWB) with gentle agitation. Washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station with gentle agitation. After washing, spin briefly in a Microarray High-Speed Centrifuge to dry.
5. Detection ith a fluorescent streptavidin secondary reagent: Prepare the secondary antibody reagent by diluting the Cy3™- or Cy5™-streptavidin conjugate to a final concentration of 1 µg/ml in Protein Microarray Reaction Buffer (Cat. PMRB). A one thousand-fold (1:1,000) dilution of the 1 mg/ml Cy3™-Streptavidin staining reagent from Jackson ImmunoResearch (Cat. 016-160-084) works well for this application. A 1.0 ml volume of secondary reagent is prepared by mixing 1.0 µl Cy3™-streptavidin in 1 ml of Protein Microarray Reaction Buffer. Mix by inverting the tube 10 times. Stain the microarrays using 25-125 µl of diluted secondary reagent. The staining volume will depends on whether a single or multi-well microarray format is being used. Stain for 60 min at room temperature with gentle agitation. Following the staining step, wash the microarrays three times for 1 min in Protein Microarray Wash Buffer (Cat. PMWB) and one time for 1 sec in Protein Microarray Rinse Buffer (Cat. PMNB) with gentle agitation. Washes can be performed using 25 ml volumes in a petri dish or 450 ml volumes in a High-Throughput Wash Station with gentle agitation. After washing, spin briefly in a Microarray High-Speed Centrifuge to dry.
6. Scan the microarrays for fluorescence emission. Scan the Microarrays using an Arrayit SpotLight 2 or Arrayit InnoScan® microarray scanner set to detect Cy3™ or Cy5™. These substrate slides can also be read with other brands of microarray scanner compatible with the open platform substrate slide 1 x 25 x 76 mm format. Scanner settings should be adjusted to minimize saturated signals to 1% or less. Multiple scans can be taken to capture data at different sensitivity levels. All data files should be saved as 16-bit TIFF images for data analysis.
Technical Specifications:
Protein A/G Microarray Substrate Slides use atomically smooth polished glass as the starting point for this product line. Users will benefit from the following technical features:
- Glass polished to atomic smoothness (±20 angstroms)
- Only polished glass surface in the microarray industry
- Polishing improves topology and uniformity – keys to precise data
- Homogeneous Protein A/G proteins provide binding to human IgG1, IgG2, IgG3, IgG4
- Protein A/G molecules are covalently bound to the glass substrate slide
- Protein A/G monolayer contains 1.1 x 10^10 proteins/mm2
- Protein A/G monolayer covers entire 25 x 76 mm surface area
- Vastly superior to unpolished optical quality glass from other vendors
- Compatible with both contact and non-contact printing
- Manufactured in a cleanroom environment
- Ultra-low intrinsic fluorescence and background noise
- Open platform dimensions compatible with all major brands of microarrayers, scanners, hybridization stations and other processing tools.
- Precise physical dimensions (0.940 mm ± 0.025 mm x 25 ± 0.2 mm x 76 ± 0.3 mm)
- Proprietary corner chamfer (1.4 mm) provides unambiguous side and end orientation to simplify printing and processing
- Finished edges enhance user safety
- Glass with excellent refractive index, transmission and hardness specifications
- Vacuum packaging improves usability
- Offered with or without barcodes
- Custom laser and chrome fiducials available upon request
- High-volume 100,000 piece per month manufacturing capabilities
- Product arrives “ready to use” with no additional processing required
Figure 2. Atomic Force Microscopy (AFM) analysis. Shown are AFM scans of the SuperClean Microarray Substrate Slide by Arrayit Corporation. Data are coded to a rainbow intensity scale, such that red data represents 5.0 nm or 50 angstroms. The substrate slides have an average smoothness of 2.0 nm or 20 angstroms, which corresponds to about 10 silicon dioxide bonds.
Figure 3. Correct Substrate Slide Orientation. Shown is a graphic of two Arrayit Microarray Substrate Slides, showing the correct and incorrect orientation for use. In the correct orientation (blue graphic), the chamfer will be located in the upper right corner and samples should be printed on the side facing upward, which is the same side that contains the word “Correct!”. In the incorrect orientation (red graphic), the chamfer will be located in the upper left corner, placing the backside facing upward, which is the side that contains the word “Incorrect!”. Only one side of Arrayit Microarray Substrate Slides is suitable for printing. Please print on the correct side only.
Recommended Equipment and Reagents
NanoPrint™ 2 Protein Microarray Printers
SpotBot® 4 Personal Protein Microarray Printers
InnoScan® Microarray Scanners
SpotLight™ 2 Microarray Scanners
Microarray Hybridization Cassettes
High Throughput Wash Stations
Microarray High-Speed Centrifuge
BlockIt™ Blocking Buffer
Microarray Air Jet
Microarray Cleanroom Wipes
Protein Microarray Buffer Kits
Green540 and Red640 Reactive Fluorescent Dyes
Hybridization Buffers