ONCYTE® Guide to Protein Microarrays - Grace Bio
Contents
1. 9 3 4 2 Film Slide Storage 9 3 4 3 Spotting Protein Concentration 9 3 4 4 Spotting Buffer 9 3 4 5 Spot Size 10 3 4 6 Spotting Controls 10 3 4 7 Spotting Environmental Conditions 11 3 4 8 Post Arraying Drying Time 11 4 Assay 11 4 1 General Methodology 11 4 2 Array Blocking2. 12 4 3 Incubation s Error Bookmark not defined 4 3 1 Buffers 13 4 3 2 Sample Concentration 13 4 3 3 Incubation Times Error Bookmark not defined 4 3 4 Incubation Chambers and Sample Mixing 14 4 3 5 Signal Amplification 17 4 4 Washing 17 5 Detection 17 6 Imaging and Data Analysis 18 Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respec
3. Maximizing Results With ONCYTE Porous Nitrocellulose Film Slides Results obtained with any microarray experiment can be highly variable if controls are not implemented during the various steps involved with the technology Figure 5 summarizes the steps required to perform a microarray experiment Variability may be introduced at any of these steps and cumulative affects can result in variability that makes results difficult to interpret Grace Bio Labs offers products which together with our premium ONCYTE film slides address significant steps in protein microarray analysis Creating protein arrays on ONCYTE film slides and using the recommendations outlined in this guide can help yield results with clear signals good spot morphology low background and low variability which allows the researcher to interpret his or her data with the utmost confidence 3 Array Printing 3 1 General Methodology ONCYTE Film Slides are ready for printing straight from the box and no activation steps are required to immobilize proteins It is important not to pre wet ONCYTE Film Slides as printing onto a wet slide will cause the sample to spread resulting in larger more diffuse spots When spotting attention to environmental conditions is critical for optimal results and temperature and humidity should be regulated In general environmental control will serve to provide more consistent results from arrays spotted during different spotting ru
4. 16 bit system Signals exceeding this limit i e saturated spots typically appear white cannot be analyzed quantitatively 6 2 Data Analysis Once image data is acquired and digitized spot intensities must be measured and analyzed There are countless methods for analyzing array data and many are customized to the experimental design employed for individual experiments It is not the goal of this guide to teach statistical methods for analyzing array data but rather to give a brief overview of the steps required to acquire measured spot intensities for subsequent analysis To this end use of a spot mask corresponding to the spotting layout is employed usually obtained from the array spotter to overlay the image file Spotfinding algorithms may be employed to fit each spot and to quantify the spot and local background intensities Typically intensities are reported in multiple ways ex mean and median pixel intensities per spot and it is commonly recommended to use the median pixel intensity as this minimizes data being skewed by noisy images ex small speckles on image It is also advisable to use local spot background measurements for performing background subtraction as opposed to taking global background measurements as these measurements may vary from location to location on an array Table 2 Laser and PMT settings for common fluorescent scanners Scanner Model Laser Power PMT Gain Axon GenePix 4100A Not Variable 400 Axon G
5. Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 1 ONCYTE Guide to Protein Microarrays June 2012 Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 2 Table of Contents 1 Introduction 4 2 Maximizing Results With ONCYTE Porous Nitrocellulose Film Slides 7 3 Array Printing 7 3 1 General Methodology 7 3 2 Contact Printing 8 3 3 Non Contact Printing 8 3 4 Important Considerations 9 3 4 1 Pre Spotting Treatment
6. Pre wetting the slides prior to spotting is not recommended and may lead to the generation of diffuse spots 3 4 2 Film Slide Storage ONCYTE film slides are specially packaged in boxes with minimal off gassing It is advised to store the film slides at room temperature in the original packaging at all times before and after printing Storage of film slides in other slide boxes may compromise results and is not recommended Many researchers store their arrayed ONCYTE film slides at 4 C or 20 C and these conditions will not harm the slides or results As a starting point it is recommended that ONCYTE Film Slides be stored overnight at 4 C after printing in order to maximize the binding of the immobilized protein before use Note It is not recommended to store printed or unprinted slides with desiccant as this may negatively impact microarray results 3 4 3 Spotting Solution Concentration Optimal source plate concentrations may vary with protein and application For capture antibodies a concentration between 250 and 1000 g ml is best for most applications However some formats may require different concentrations For cell tissue lysates the highest protein concentration possible is usually desired to detect rare antigens Typically serial dilutions 1 1 1 2 1 4 etc are spotted in parallel to establish that the assay is in linear range of detection The assumption is that the target on the slide should not be limited in concentr
7. by their respective owner 19 may require larger spot sizes if results are to be quantified For optimum use of the dynamic range of the scanning system it is recommended to save data using the maximum depth compatible with the evaluation software e g 16 bit tiff file 6 1 2 Scanner Settings When imaging ONCYTE Film Slides for fluorescent applications the default imager parameters for glass slides will not be suitable for detection Due to the higher binding capacity of ONCYTE Film Slides as well as the unique light scattering properties of the polymeric surface laser power and or PMT settings voltage gain will need to be set lower than for glass slides If the scanner has confocal optics and focal depth adjustment the focal depth should be optimized since the nitrocellulose coating is approximately 12 m thick On non confocal systems this is not necessary as the fixed focal depth of field is usually larger than the thickness of the NC layer e g the Axon GenePix 4100A has 40 m depth of field The laser and PMT settings will also depend on the type of experiment and blocking agent used Typical starting parameters for some popular instruments are given in Table 2 For best results these settings should be further optimized In order to take full advantage of the dynamic range of the scanner signal intensities should be as high as possible without reaching or exceeding the maximum of the system i e pixel intensity 65535 on a
8. detection taking into consideration the possibility of lateral bleeding of signal onto substrates such as X ray film A consideration to keep in mind for quantitative analysis of array images regardless of the detection method is that the pixel size should be no more than 1 10th of the spot diameter i e at least 10 pixels across the spot s diameter Choice of appropriate spotting buffer additives arrayer pins and proper environmental control during array spotting are all critical parameters which can be used to attenuate the spot size to the desired level 3 4 6 Spotting Controls As previously mentioned microarray experiments are subject to many sources of variation which can be introduced during array spotting A key consideration which allows for assessment of array spotting quality and is also useful during data analysis for normalization if required is the choice of appropriate spotting controls Spotting controls will allow the researcher to identify poor spotting runs and poor protein binding to the array and are also useful for normalizing experimental data and troubleshooting during assay development An example of a spotting control would be IgG pre labeled with a fluorophore and spotted at a known concentration These control spots should be distributed equally at different coordinates of the arrayed surface alongside the regular array content Some researchers choose to include a pre labeled control in each sample well Re
9. in small volume chambers and in some cases under coverslips In general use of a staining jar coupled with an orbital shaker allows for adequate sample mixing and is an excellent choice for blocking and wash steps where volume is not a limiting factor In many cases though the primary binding reaction incubation steps of the microarray assay are volume limited due to the availability of probe In these cases use of larger containers such as staining jars is not feasible due to excessive sample dilution Smaller chambers are preferred for these steps Coverslips generally allow for the lowest sample incubation volumes and although not ideal due to limited sample mixing by diffusion some users are driven by limited sample volumes Kersten et al 2003 Another disadvantage of using coverslips is the potential for damaging the array surface when manually removing the coverslip If the use of a coverslip is necessary for your particular assay we recommend the use of Grace Bio Labs HybriSlip over conventional glass coverslips Figure 7 In addition Grace Bio Labs has developed incubation chambers which facilitate incubations for a wide range of sample volumes with various levels of mixing In particular the Pro Plate chamber from Grace Bio labs www gracebio com is very well suited for most protein array applications when using ONCYTE film slides ProPlate incubation chambers are available in various formats corresponding to available
10. of this section 3 2 Contact Printing Contact printing utilizes pin type arrayers that transfer a defined volume of sample by directly touching the surface of the slide Despite the relatively soft nitrocellulose surface of ONCYTE Film Slides contact printing can be performed without physically damaging the coating if the arrayer settings are appropriately adjusted It is recommended to use contact arraying systems that feature free floating pins in their print heads as opposed to spring loaded pin mechanisms Contact printers are usually simpler in design less expensive and faster than non contact printers depending on pin configuration and they may be the best choice when large numbers of samples or highly viscous samples will be spotted such as with RPPAs Split pins quill type pins and solid pins have all been successfully used for printing proteins on ONCYTE Film Slides Some pin cleaning protocols recommended by manufacturers have been optimized for DNA printing applications and may not be best for spotting of proteins because of higher viscosity and adhesiveness of proteins compared to nucleic acids Addition of a surfactant such as Bioterge AS 40 in a very low concentration e g 0 025 to the wash solution has been found to be advantageous when using quill pins Ring and pin printers are a variant of contact printers Samples are taken up from the source plate with rings mounted in front of the spotting pins The pin passes the
11. 22 10 Misaligned spots If contact printing bent or broken pin s If non contact arraying arrayer tip is misfiring Contact printer check integrity of pins under microscope adjust position replace bent broken pin s Non contact printer check tip status settings 11 Donut shaped spots Strike force of pin on surface too high Viscosity of sample very high Humidity too low during spotting Reduce strike force Add glycerol to source solution 5 to 10 Increase humidity of printing chamber Donut effect cannot be completely avoided if spot finding in the data reduction software is performed properly it has little effect on the results 12 Comets and Tadpoles Loosely bound capture protein moves during sample incubationhybridization Wet slide surface during printing see above Protein arrayed using too high concentration Prolong drying time after printing gt overnight Do not use cold out of the fridge slides risk of condensation water on surface Reduce concentration of capture protein 13 Speckled background Particles in solution Replace with fresh particle free solvents Make sure that no precipitate is in sample Centrifuge sample before incubation Filter sample solvents through 0 45 m syringe filter 8 Further Information The referen
12. ONCYTE film slide configurations from single well to 64 wells Figure 8 ProPlate chambers enable thorough mixing of samples during incubations with the use of an orbital shaker Active mixing with these devices has been shown to significantly decrease assay variation within spots across spots within individual array slides and across spots on multiple array slides when compared to coverslip incubations Figure 9 The effect of this mixing is more robust microarray data In addition ProPlate chambers can simplify the microarray workflow as they are compatible with multi channel pipettes and allow for convenient sample dispensing and buffer replacement during solution changes HybriSlip HybriWell Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 15 Figure 7 From Grace Bio labs See www gracebio com Figure 8 ProPlate chambers enable active mixing of sample during hybridization The chambers attach to slides for hybridization and are removed for imaging If using ProPlate chambers the blocking step may be performed across the entire slide prior to attachment of the chamber or may be performed in individual chambers Appropriate volumes for the various chamber configurations are listed in Table 1 For blocking and washing steps the maximum volumes are recommended Incubate at appropriate temperature with ge
13. Paweletz C P et al 2001 Reverse phase protein microarrays which capture disease progression show activation of pro survival pathways at the cancer invasion front Oncogene 20 1981 1989 16 Sakanyan V 2005 High throughput and multiplexed protein array technology protein DNA and protein protein interactions J Chromatography B 815 77 95 17 Schweitzer B et al 2003 Microarrays to characterize protein interactions on a whole proteome scale Proteomics 3 2190 2199 18 Spector N L et al 1994 Regulation of the 28 kDa heat shock protein by retinoic acid during differentiation of human leukemic HL 60 cells FEBS Lett 337 184 188 19 Yeretssian G et al 2005 Competition on nitrocellulose immobilized antibody arrays From bacterial protein binding assay to protein profiling in breast cancer cells Mol Cell Proteomics 4 605 617 20 Zhou H et al 2004 Two color rolling circle amplification on antibody microarrays for sensitive multiplexed serum profile measurements Genome Biology 5 4 R28 21 McGrath C et al 1991 Coherent Transfer of Receptor Proteins on Microporous Membranes BioTechniques 11 352 361 22 Kingsmore SF 2006 Multiplexed protein measurement technologies and applications of protein and antibody arrays Nat Rev Drug Discov 5 4 310 20 Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by t
14. ation relative to the sample probe 3 4 4 Spotting Buffer Protein arrays may be designed with many types of targets antibodies antigens purified proteins or complex cell lysates or protein mixtures may be deposited on the array For applications where the native conformation of the deposited proteins is desired an arraying solvent must be chosen that maintains the protein s molecular structure and or recognition properties Non denaturing spotting solutions should consist of a buffer with suitable pH and ionic strength and may contain other stabilizing agents like protease inhibitors chelators etc PBS is often a suitable spotting solution Addition of non denaturing detergents e g 0 1 Tween 20 may help with controlling spot size and morphology Some applications may require the presence of detergents and or chaotropes e g urea for cell disruption and or solubilization of proteins Generally substances like these are compatible with ONCYTE Film Slides Solubilization buffers originally designed for 1D and 2D electrophoresis Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 10 are compatible with nitrocellulose film slides For example a buffer containing 6 M urea and 2 CHAPS has been successfully applied for array printing Nishizuka et al 2003 SDS containing buffers also perform well and exhibit very efficient prot
15. ay Blocking Buffer for 1 hour prior to assay B Slide not blocked soaked in PBS for 1 hour prior to assay A B Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 13 basis A good starting point is 1 BSA e g Sigma cat no A 7638 cold alcohol precipitation fractionation prepared from Fraction V bovine albumin 0 05 Tween 20 in PBS pH 7 2 7 5 Casein based solutions are efficient blockers due to a broad spectrum of molecules of different sizes However because of poor solubility casein based solutions carry the risk of causing speckles on the arrays due to precipitates 4 3 Binding Assay In many applications pure capture molecules antibodies recombinant proteins are immobilized on the slide surface Generally this array is incubated with a complex solution to be analyzed for the presence and concentration of specific binding partners To allow for and detect this interaction one or more incubations will be performed after blocking and may vary from assay to assay All conditions and parameters will require optimization to obtain the highest quality data Key parameters which should be assessed are discussed in the remainder of this section 4 3 1 Assay Buffer In order to define a suitable hybridization buffer for the probe the same general considerations apply as for the printing buffer The assay buffer mu
16. ce Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 12 4 2 Blocking Blocking a microarray surface helps to reduce non specific binding of probe molecules or dyes Blocking may be performed in bulk solution utilizing a histology staining jar e g Coplin Corning Wheaton Place arrays in a slide cradle and immerse in blocking solution typically 200 ml in a Wheaton staining jar Blocking can be performed with or without shaking on an orbital shaker Blocking time will vary and should be determined for your applications Depending on the sample blocking times can range from 15 min to overnight and need to be determined empirically For long incubation times i e hours care must be taken that the slides do not dry out The use of a humid chamber e g a zip lock bag with wetted paper towel is highly advisable Proper blocking is imperative for obtaining the best signal to noise ratio possible from any array experiment The blocking step should be performed after slides thoroughly dried after printing typically overnight at 4 C and the type of blocker used will depend on the nature of the experiment Grace Bio Labs Super G Protein Array Blocking Buffer was developed and optimized for use with ONCYTE Film Slides This blocking reagent was primarily developed for use in fluorescent assays but is also compatible with other common detection methods It is recommende
17. ces below include several articles that give a review of specific fields of application of protein microarrays Espina et al 2003 review the use of reverse phase arrays in cancer research Array platforms for analysis of autoimmune diseases are reviewed by Balboni et al 2006 A review of microarrays for glycosylation research was written by Feizi and Chai 2004 Sakanyan 2005 and Schweitzer et al 2003 review the use of protein arrays to study protein protein interaction A general review on proteomic studies using microarrays was written by Feilner et al 2004 Additional information can be found on the Grace Bio Labs web pages www gracebio com Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 23 9 References 1 Balboni I et al 2006 Multiplexed protein array platforms for analysis of autoimmune diseases Annu Rev Immunol 24 391 418 2 Calvert V S et al 2004 Development of multiplexed protein profiling and detection using near infrared detection of reverse phase protein microarrays Clin Proteomics 1 81 89 3 Ekins R and Chu F 2003 Ultrasensitive microarray based ligand assay technology In Protein Arrays Biochips and Proteomics The next Phase of Genomic Discovery Albala J S and Humphrey Smith I Eds Marcel Drecker Inc New York Basel PP 81 125 4 Espejo A et al 2002 A protei
18. d for its superior blocking power and the resulting low non specific binding background producing superior signal to noise Grace Bio Labs has spent decades optimizing the production of our ONCYTE nitrocellulose films to minimize the inherent background so commonly associated with nitrocellulose film slides We have found that for fluorescent assays incomplete blocking of slide surface prior to hybridization will reduce or eliminate the advantages of our premium films Optimal performance of our ONCYTE films can only be guaranteed with use of Super G Protein Array Blocking Buffer Many researchers utilize blocking protocols identical to those used with Western Blots on nitrocellulose membranes A physiological buffer 1x PBS or 1x TBS containing 1 5 non fat milk is compatible with isotopic and chemiluminescent detection For fluorescent detection 1x TBS containing 0 1 Tween 20 1x TBS T may be sufficient for some applications The percentage of Tween 20 may be increased to 2 if needed Preliminary experiments should be conducted to determine the optimal blocker and concentration for fluorescent systems Some blockers can add to the fluorescent background and should be chosen carefully For this reason blocking buffers containing protein such as BSA casein or non fat dry milk should be examined on an empirical Figure 6 Effects of proper blocking on ONCYTE film slides A Slide blocked with Grace Bio Labs Super G Protein Arr
19. e Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 6 Protein arrays can be designed in a number of different configurations which can be used in a wide variety of downstream assays In Forward Phase Protein Arrays antibodies are arrayed as capture molecules and used to perform quantitative profiling of protein expression or for detecting the presence of their antigens in complex lysates after direct or hapten labeling Figure 4A and 4B In other configurations recombinant or purified proteins can be immobilized to study protein protein interaction or to probe sera for the presence of specific antibodies Figure 4C and 4D Another protein array configuration gaining increasing attention is the Reverse Phase Protein Array RPPA where complex tissue or cell lysates from tissues taken under varying conditions e g dose response experiment or from patient samples e g different tumor types are immobilized These samples are probed with an antibody for the antigen of interest in order to profile the presence of this antigen Figure 4E Figure 4 Design of Protein Microarrays Depicted above are some commonly employed configurations A Antibodies used to capture specific antigens which are directly labeled with a hapten or B for detection in ELISA like sandwich assays C Purified or recombinant proteins can be arrayed to study protein protein interaction or D to probe
20. ein binding for applications not requiring proteins in their native conformation Highly viscous spotting solutions may give poor spotting results largely due to limitations of the printers and clogging of the pins or print jets DMSO is often added to printing buffers to reduce evaporation of the solution resulting in variable concentration of sample during the printing process The use of DMSO for ONCTYE Film Slides is generally not recommended because high concentrations of DMSO can negatively affect the nitrocellulose If DMSO is added to the printing buffer final concentrations should not exceed 5 3 4 5 Spot Size A parameter closely tied to the choice of spotting buffer is the desired required spot size Smaller spots provide higher analyte density and typically better signal to noise ratios Ekins and Chu 2003 Technical constraints for spot size can come from protein concentration and or viscosity of the sample that may dictate the choice of printing system and hence may place constraints on the achievable spot size The resolution of the detection system is another consideration which may require the optimization of spot size For example isotopic detection autoradiography on x ray film or image phosphor screens and chemiluminescence have generally much lower spatial resolution than colorimetric or fluorescence detectors CCD camera or scanner systems Additionally the spot pitch should be large enough to avoid spot overlap during
21. enePix 4200A 95 400 Tecan LS200 Not Variable 95 Perkin Elmer ScanArray 4000 80 40 Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 20 7 Troubleshooting Fluor fluorophore Symptom Cause Remedy 1 Low Signal Scanning PMT laser power too low Sample no binding sample concentration too low Detection antibody does not bind Fluor conjugate concentration too low Increase PMT laser power of scanner Use more concentrated sample Include suitable positive controls Increase fluor conjugate concentration 2 Saturated spots White coloring indicates spot saturation avoid saturated spots cannot be quantified PMT laser power too high Reduce PMT laser settings 3 High background and saturated spots White coloring indicates spot saturation Avoid saturated spots cannot be quantified PMT laser power too high Brightness contrast in imaging software not set appropriately Insufficient blocking Reduce PMT laser settings Reduce brightness increase contrast Background cannot be judged visually measure background with analysis software and assess signal to noise refer to Symptom 4 Prolong blocking time gt 30 min overnight optimize blocking b
22. eps each separated by washing steps Incubation times for the samples being tested are generally the longest step in the protocol from one hour to overnight Hence care must be taken to prevent the arrays from drying out The use of a humid chamber is highly advisable zip lock bag or similar If the sample is directly labeled with a fluorophore protection from light during incubation is also Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 14 recommended 4 3 4 Incubation Chambers and Sample Mixing The incubation chamber is an important parameter which is often overlooked when performing microarray experiments Arrays may be processed in various types of chambers the choice of which can significantly affect the quality of the microarray data generated For many users the choice of chamber will be dependent on one primary factor the volume of sample available for the assay Regardless of the chamber chosen though it ideally should allow for sufficient sample mixing during the assay incubation and wash steps Adequate mixing has been shown to significantly affect assay signal and uniformity When ample quantity of probe is available arrays may be processed in bulk solution using incubation chambers such as slide mailers and staining jars Alternatively if probe protein is limited in quantity hybridization may be performed
23. esponding Alexa and Dyelight fluorophores Phycoerythrin and others Infra red fluorophores such as IR800 have also been used with excellent results Calvert et al 2004 Yeretssian et al 2005 In general it has been observed that longer wavelength fluorophores such as Cy5 and analogs or IR800 are often advantageous Many biomolecules present in blocking reagents and samples have an inherent autofluorescence and will bind to the surface thus contributing to background Blocking with Super G will significantly minimize background fluorescence but this phenomenon can be further lessened when using red and far red wavelengths for detection Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 18 6 Imaging and Data Analysis 6 1 Imaging For preparation of slides for chemiluminescent colorimetric and isotopic applications follow standard nitrocellulose membrane protocols for the specific detection reagents being used In some instances e g chemiluminescence and some staining protocols the slide will remain wet and can be placed in plastic wrap or other material to prevent drying For fluorescent detection using a microarray based imager the slide should be dried after the final wash Excess water droplets on the edges can be removed gently with a lint free tissue or a compressed N2 stream Be careful not to damage the surface as this
24. gardless of the method of choice appropriate for your application Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 11 including spotting controls merits attention early in the process of your assay development It is also important to deposit replicates for each protein spotted on the array As previously stated microarray experiments are subject to a variety of sources of noise and include artifacts caused by speckles precipitates or dust particles either in the air or in the spotting buffer By including replicates a researcher can take the representative value e g median geometric mean from multiple spots so that one faulty spot does not significantly impact the experimental results 3 4 7 Spotting Environmental Conditions Temperature and humidity should be regulated to control spot drying as well as to avoid evaporation of source plate solutions during the arraying process and in the case of contact printing to avoid evaporation of sample from spotting pins during pin travel In general too low humidity may cause spots to dry too quickly causing spots with higher protein concentration on the spot perimeter typically manifesting as donut spots Too high humidity may cause larger spots and potentially spot to spot bleeding depending on spot pitch Additionally high humidity may lead to problems with water conden
25. h either HRP or AP either chemiluminescent or chromogenic substrates can be used If using AP based chemiluminescent substrates enhancers for nitrocellulose may be required depending on the reagent system employed 4 4 Washing A physiological buffer should be used for washing that preserves the protein protein or protein nucleic acid interaction yet washes away unbound sample A good wash buffer is generally the same as the blocking solution used minus the blocking agent As a starting point a typical wash protocol consists of 3 washes with PBST 5 min each with shaking followed by 3 washes with PBS 5 min each with shaking followed by a 1 min rinse with filtered water at room temperature Slides are then dried with a stream of dry N2 or by centrifugation prior to detection 5 Detection ONCYTE Film Slides are compatible with many detection methods Figure 10 Common methods employed routinely for Western and Northern blots are compatible with porous nitrocellulose films and include fluorescent chemiluminescent isotopic and chromogenic detection The method of choice used for protein detection will depend on the application For example for analysis of phosphorylation labeling with 32P ATP and detection by autoradiography is still considered a very reliable method Detection using fluorescent dyes is very convenient has high spatial resolution as well as very high sensitivity Commonly used fluorophores include Cy3 Cy5 corr
26. heir respective owner 24 23 Van Oss CJ et al 1987 Mechanism of DNA Southern and protein Western blotting on cellulose nitrate and other membranes J Chromatography 391 1 53 65 24 Tang Y et al 2003 Blood genomic expression profile for neuronal injury J Cereb Blood Flow Metab 23 3 310 9 25 Stillman BA and Tonkinson JL 2000 FAST slides a novel surface for microarrays Biotechniques 2000 Sep 29 3 630 5 26 Oh SJ et al 2000 Surface modification for DNA and protein microarrays OMICS 2006 Fall 10 3 327 43 27 Espina V et al 2003 Protein microarrays molecular profiling technologies for clinical specimens Proteomics 3 11 2091 100
27. may lead to background artifacts A simple drying method is to spin the slides briefly using a 50 ml conical tube as a slide holder in a suitable centrifuge 2 3 minutes at 150 x g followed by at least 10 minutes of drying at room temperature in a dust free dark place until imaging If stored as recommended the signals remain stable over weeks and months after processing 6 1 1 Imaging Instruments and Image Resolution ONCYTE Film Slides can be analyzed using a variety of laser scanners or CCD imaging systems As a general rule of thumb spot diameter should be at least 10x pixel size in order to sample sufficient data for a quantitative analysis For arrays printed according to the recommended settings a resolution of 10 m will be optimal Imaging instruments such as gel imagers usually work at considerably lower resolutions pixel size 25 m or larger Thus other imaging systems Figure 10 Examples of results obtained with ONCYTE film slides with various detection methods A Typical result obtained with colorimetric endpoint courtesy of Satoshi Nishizuka Iwate Medical University B typical result obtained with fluorescent endpoint detection courtesy of an of OEM partner and C typical results obtained with IR dye fluorescent endpoint detection courtesy of University of Nottingham Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered
28. n domain microarray identifies novel protein protein interactions Biochem J 367 697 702 5 Espejo A Bedford M T 2004 Protein domain microarrays Methods Mol Biol 264 173 184 6 Feilner T et al 2004 Proteomic studies using microarrays Current Proteomics 1 4 293 295 7 Feizi T and Chai W 2004 Oligosaccharide microarrays to decipher the glyco code Nature Reviews Mol Cell Biol 5 582 588 8 Gutjahr C et al 2005 Mouse protein arrays from a Th1 cell cDNA library for antibody screening and serum profiling Genomics 85 285 296 9 Kim T E et al 2002 Quantitative measurement of serum allergen specific IgE on protein chip Exp Mol Med 34 2 152 158 10 Kersten B et al 2003 Generation of Arabidopsis protein chips for antibody serum screening Plant Mol Biol 52 999 1010 11 Kukar T et al 2002 Protein microarrays to detect protein protein interactions using Red and Green Fluorescent Proteins Anal Biochem 306 50 54 12 Lueking A et al 2005 Profiling of Alopecia areata autoantigens based on protein microarray technology Mol Cell Proteomics 4 9 1382 1390 13 Madoz G rpide J et al 2001 Protein based microarrays a tool for probing the proteome of cancer cells and tissues Proteomics 1 1279 1287 14 Nishizuka S et al 2003 Proteomic profiling of the NCI 60 cancer cell lines using new high density reverse phase lysate microarrays PNAS 100 24 14229 14234 15
29. ns Pure proteins should be arrayed using a source plate concentration of 0 05 1 mg ml A concentration between 50 and 1000 g ml is optimal for most applications For antibodies the upper end of this range is recommended If using a fluorescent scanner for subsequent detection a spot diameter of 250 m or less is recommended and the array pitch distance of neighboring spots from center to center can be as low as 300 m For chemiluminescent or isotopic detection spot Figure 5 General outline of a typical microarray experiment Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 8 diameter can be significantly larger than for fluorescent detection However a pitch of 1000 m or greater is recommended to allow for sufficient resolution Different microarray printers come with their own control software which can vary significantly in look and feel All robotic printers accommodate a specific format of the input source material typically a 96 or 384 well plate the desired arraying pattern on the slide surface the cleaning parameters between sample wells and depending on the spotter control of environmental conditions during spotting All of these parameters will vary depending on the individual applications and in many cases require some degree of optimization Key parameters are discussed in more detail in the remainder
30. ntle agitation on an orbital shaker ensuring that mixing occurs See product insert for complete instructions or visit www gracebio com Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 16 Based on histology staining methods some researchers simply cover the array with liquid kept in place by encircling the array with a hydrophobic marker pen e g PAP pen Kukar et al 2002 This method suffers the same disadvantages as with coverslips limited sample mixing In addition this and any method utilizing an open chamber allows evaporation and drying of the array and subsequent concentration of the hybridization solution may occur even for short incubations We recommend the use of humidified chambers to minimize this effect Table 1 Suitable incubation volumes for use with ProPlate chambers ProPlate Format Well Volume Min Max 1 Pad 700 2000 l 2 Pad 250 1000 l 4 Pad 125 500 l 8 Pad 70 250 l 16 Pad 70 125 l 64 Pad 20 50 l For users with access to automated systems single pad ONCYTE Film Slides may be processed with devices designed for processing of immunohistochemistry slides Paweletz et al 2001 or automated hybridization chambers e g GeneTAC Hybridization Station Digilab Genomic Solutions Inc for processing of DNA microarrays Madoz G r
31. pads dried out during incubations Reduce speed of shaker to 40 rpm Use humid chamber for long incubations Cover ProPlate with lid if condensation of water is observed under cover place piece of polystyrene foam under frame for insulation from heat generated by shaking instrument 7 Black holes spot appears darker than surrounding background Arrayed probe does not bind sample but shows blocking effect In many cases this is a normal effect Change buffer for the proteins arrayed i e remove additives that lead to blocking effects 8 Scratches Surface of slide touched with pipette tip Always take care not to touch the surface of ONCYTE Slides always remove aspirate liquid from corners of pads 9 Missing spots Bent or broken pin s Salt other material on tip of pin s Clogged pin s Volume difference in source plate Wet slide surface Check integrity of pins under microscope adjust position replace bent broken pin s Clean pins thoroughly after each printing run optimize washing protocol see manual of printing instrument Check source plate Do not use cold out of the fridge slides risk of condensation water on surface Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner
32. pide et al 2001 Automation can improve reproducibility of results by eliminating variability in processing times and Figure 9 Effects of active mixing Microarray slides were fitted with a ProPlate chamber during assay incubations Coefficient of variation is compared against results obtained from an identical assay under a cover slip where mixing is limited to sample diffusion All data obtained from spotting concentration of 1 mg ml within the linear dynamic range of detection Fluorescence data are normalized and background subtracted collected at 532 nm from spotted goat IgG assayed with rabbit anti goat IgG TRITC CoverSlip 50 l 1 25 000 Dilution 4 well ProPlate 300 well 1 150 000 Dilution Intra Spot CV data are pixel variation per spot N 64 spot replicates over 4 microarrays Intra Slide CV data are spot variation per array N 4 microarrays mean of N 16 spot replicates array Inter Slide CV are for an N 4 microarray slide replicates Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 17 temperatures The automated system must accommodate the full coated area of the slide 4 3 5 Signal Amplification For some applications signal amplification will be necessary when antigen levels are very low Frequently systems employing horseradish peroxidase HRP and alkaline phosphatase AP are used Wit
33. rge constituency of molecules in parallel utilizing minimal sample and reagents While many instruments and software tools are shared for all applications the surface and labeling chemistries may vary depending on the molecules of interest whether nucleic acids proteins inorganics or organics A variety of surface chemistries are available with new chemistries emerging every year However nitrocellulose has remained a favored substrate for protein microarray applications due to its many functional advantages and most importantly its high binding capacity for protein Grace Bio Labs developed its first nitrocellulose film slide in 1990 McGrath et al 1991 and continues to produce film slides with the highest protein binding capacity lowest inherent auto fluorescence and best therefore signal to noise compared to other nitrocellulose film slides Nitrocellulose films have a long history of use in Western Northern and dot immuno blots for reliable immobilization and capture of biomolecules They have also been used in the manufacture of lateral flow immunoassays such as pregnancy tests in the diagnostic industry The 3 dimensional structure of ONCYTE Nitrocellulose Film Slides offers a considerably higher surface area for protein binding compared to conventional 2 dimensional surfaces Figure 1A and 1B Increased surface area for binding translates into increased binding capacity in microarray spots and is related to pore size pore str
34. ring to deposit sample on the slide surface This technology has been successfully used with very viscous tissue lysates containing high concentrations of urea and detergents Nishizuka et al 2003 Examples of contact printers that have been successfully used for array production on ONCYTE Film Slides include Aushon 2470 SpotBot and NanoPrint ArrayIT Corporation OmniGrid and MicroGrid Genomics Solutions Ltd and Q array Genetix Ltd 3 3 Non Contact Printing Non contact printers can be syringe based solenoid or piezo type With these technologies sample droplets are dispensed onto the slide avoiding contact of the print pen with the surface Sample volume can be varied in steps by firing multiple times on the same spot This technique enables very high reproducibility and speed when manufacturing large numbers of limited content arrays As with contact deposition printers pen cleaning protocols should be optimized for protein samples Examples of systems which have been successfully used with ONCYTE Film Slides are the Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 9 NanoPlotter GeSiM and sciFLEXARRAYER Scienion AG 3 4 Important Considerations 3 4 1 Pre Spotting Treatment ONCYTE film slides require no pre processing prior to array spotting They are ready for spotting right out of the package
35. sation in the spotter Refer to the manual of the arraying system 3 4 8 Post Arraying Drying Time Stability of protein binding to the nitrocellulose matrix has been found to increase with appropriate drying time As a starting point it is recommended to store the spotted array overnight at 4 C prior to use to allow optimal binding of the printed proteins As with protein concentration this parameter should be optimized for your particular proteins and assay Long term storage of spotted arrays should also be optimized and can be performed at 4 C or 20 C 4 Assay 4 1 General Methodology The sequence of steps required for a microarray experiment will vary depending on the application and detection methodology utilized Most microarray assays will first employ an array blocking step to inactivate any unbound portions of the array surface Blocking is usually followed by a series of wash steps aimed to remove any unbound spotting material The array is then ready for incubation with a primary antibody cell tissue extract serum or probe molecule Multiple wash steps may follow or further incubations with a secondary antibody and or other probes Depending on the detection method there may be multiple cycles of binding washing with the ultimate incorporation of a detection molecule or amplification system Typically arrays are washed and dried at the completion of all incubations and prior to detection Grace Bio Labs 022012 2011 Gra
36. serum samples for antibodies E Reverse Phase Protein Arrays RPPA are used to profile dozens or hundreds of arrayed samples e g cell or tissue lysates for the presence of selected antigens In actual practice the non covalent bonding of proteins to nitrocellulose is not reversible under normal Figure 4 Protein binding capacity for ONCYTE AVID compared to 2 dimensional nitrocellulose Gentel PATH and aminosilane functionalized glass slides The ONCYTE 3 dimensional surface allows for approximately 500 times the protein binding capacity Data are normalized background subtracted fluorescence intensities collected at 532 nm from spotted goat IgG Cy3 Data presented are the mean standard deviation for N 4 slides per slide type 20 spot replicates per slide Figure 3 Dynamic range of protein binding for ONCYTE AVID film slides spans over 7 orders of magnitude with a linear range of 6 orders of magnitude r2 0 999 Data are normalized background subtracted fluorescence intensities collected at 532 nm from spotted goat IgG Cy3 Data presented are the mean standard deviation for N 4 slides 20 spot replicates per spotting concentration per slide Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 7 spotting and assay conditions used in microarray applications Stillman et al 2000 Oh et al 2006 2
37. st allow proteins to maintain their molecular biologically relevant structure and recognition properties In addition to stabilizing agents it is recommended to include a blocking agent in the assay buffer An appropriate buffer for sample incubation can be PBS pH 7 2 7 5 containing 0 05 Tween 20 0 1 BSA and including other additives such as protease inhibitors This may also serve as a generic dilution buffer if samples are to be diluted 4 3 2 Sample Concentration Protein concentrations in the probe sample will vary based on the nature of the experiment They will be governed both by concentration of the molecules to be measured and affinity of the arrayed antibodies for them Hence individual assay conditions must be determined empirically Different sample dilutions e g 1 2 1 4 should be tested 4 3 3 Incubation Times The incubation time needed to establish equilibrium binding will vary with the type of experiment If probing with an antibody in an experiment analogous to a Western Blot 1 2 hours at room temperature are usually sufficient Other types of samples may need to be incubated overnight in order to maximize intensity of interaction Assay incubations longer than 24 hours may provide diminishing returns as non specific binding will continue to increase after specific binding is saturated Optimal conditions should be determined empirically Depending on assay configuration there may be 2 or more separate incubation st
38. tive owner 3 6 1 Imaging 18 6 1 1 Imaging Instruments and Image Resolution 18 6 1 2 Scanner Settings 19 6 2 Data Analysis 19 7 Troubleshooting 20 8 Further Information 22 9 References 23 Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 4 1 Introduction Technology advances in instrumentation chemistry and software analysis have expanded the use of microarrays and greatly improved the reliability and performance over the past few decades Microarrays offer the ability to analyze a la
39. ucture pore density and film thickness For example ONCYTE AVID porous nitrocellulose slides show up to 500 times the binding capacity of conventional 2 dimensional surfaces Figure 2 Coupled with low fluorescence background the higher binding capacity of these slides provides a very broad linear dynamic range for detection up to 7 orders of magnitude Figure 3 This is especially important for the development of quantitative protein microarray assays for research and diagnostic applications Nitrocellulose films are particularly well suited for reverse phase protein arrays RPPA see figure 4E where maximizing the amount of spotted protein is critical to the experimental outcome Paweletz et al 2001 Balboni et al 2006 The advantages of nitrocellulose also stem from the nature of the protein to matrix interaction which allows for retention of three dimensional structure and function of the bound material figure 1C Binding of biomolecules to nitrocellulose occurs through combined weak intermolecular forces probably dominated by hydrophobic and van der Waals forces Van Oss et al 1987 Tang et al 2003 Kingsmore et al 2006 Importantly retention of protein structure is required for many antibody interactions often used for detection of proteins in microarrays A B C Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective o
40. uffer see section 4 2 4 High background and weak signals Insufficient blocking Matrix effect of sample Direct sample labeling unbound dye in solution protein concentration too high Indirect labeling fluor conjugate concentration too high Prolong blocking time gt 30 min overnight optimize blocking buffer see section 4 2 Use Super G blocking buffer Dilute sample Direct sample labeling remove unbound dye use spin column Indirect labeling reduce fluor conjugate concentration Grace Bio Labs 022012 2011 Grace Bio Labs Inc All rights reserved All the trademarks or brands in this document are registered by their respective owner 21 5 Cloudy background Insufficient washing Dry out of slide during processing Final wash step water rinse left out or not long enough Post processing slide drying Protocol not followed correctly Use more wash steps minimum 3 changes Increase detergent concentration in wash buffer Tween 20 up to 2 Increase wash temperature 37 C Never let slide dry out during processing Use humid chamber for all incubation steps longer than just a few minutes Work as quick as possible when changing solutions use multichannel pipette 6 Swirls or smeary stripes Vortexes and wave pattern forming during shaking with sample Portion of
41. wner 5 Figure 1 A Scanning electron micrograph 20 000x magnification showing the three dimensional structure of ONCYTE films B Depiction emphasizing the advantages to spotting on a 3 dimenstional ONCYTE matrix Scanning electron micrograph 2 000x magnification of an ONCYTE film and a depiction of the volume a spot would fill through the thickness of the 3 D film left compared to a depiction of the same spot on a two dimensional surface right C Depiction of the three dimensional surface of an ONCYTE film which allows for the retention of the three dimensional structure and function of spotted protein In this case DNA polymerase is depicted in a form which retains its structure thus allowing its use in a functional assay on the solid support of an ONCYTE slide C B A T ype a quo te fro m the doc um ent or the sum mar y of an inte rest ing poi nt You can posi B B A B C Figure 2 Protein binding capacity for ONCYTE AVID compared to 2 dimensional nitrocellulose Gentel PATH and aminosilane functionalized glass slides The ONCYTE 3 dimensional surface allows for approximately 500 times the protein binding capacity Data are normalized background subtracted fluorescence intensities collected at 532 nm from spotted goat IgG Cy3 Data presented are the mean standard deviation for N 4 slides per slide type 20 spot replicates per slide Grace Bio Labs 022012 2011 Grac
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