User Manual ProtoArray® Immune Response Biomarker Profiling
Contents
1. Apply the sample on the side with the raised edge If unsure of the raised side draw the point of a pipette tip across the edge the rough side is the side with the edge Raised edges Pipette 100 ul diluted human serum sample dilute the sample 1 150 in Probing Buffer onto the inverted LifterSlip in a line at the center of the slip Avoid pipetting any bubbles Using the same pipette tip draw the solution across the entire length and width of the LifterSlip such that the serum sample is spread as an oval shape and is within 2 mm from the edges of the LifterSlip without any dry spots on the center After removing the array from the blocking chamber briefly dry the back side and edges of the array using an absorbent laboratory wipe Place the array horizontally in a chamber such as a petri dish or tray with the printed side facing up Note The incubation step with the serum is performed in a covered chamber such as a petri dish or tray and not the Array Chamber supplied with the kit to obtain the best results The LifterSlip tends to come off when the array is placed vertically in the Array Chamber Carefully lift the LifterSlip cover slip with the sample and quickly invert the cover slip in one smooth motion such that the sample is on the underside of the cover slip The sample is held on the cover slip due to surface tension and does not fall Continued on next page Performing Serum Profiling2. Array Chambers included in the ProtoArray IRBP Room Temperature Module and LifterSlips included in the ProtoArray IRBP Room Temperature Module Make sure the buffers are cold Store buffers on ice until use Place the Array Chambers on ice to chill the chamber until use Review Important Guidelines on page 7 prior to starting the probing procedure Two methods are suited for blocking the arrays and is dependent on the number of arrays that you wish to process Blocking in mailer is performed when you wish to process a small number of arrays 4 arrays The mailer is the container in which the array is supplied To block using the mailer you need 30 ml buffer per mailer Blocking in glass slide chambers available from VWR catalog no 74280 014 is performed when you wish to process large number of arrays gt 4 arrays To block using the glass slide chamber you need 120 ml buffer per chamber Continued on next page Performing Serum Profiling Continued Blocking Step Immediately place the mailer containing the ProtoArray Human Protein Microarray nc v4 0 at 4 C upon removal from storage Equilibrate the mailer at 4 C for at least 15 minutes prior to performing the blocking step Perform blocking as described choose an appropriate method based on the number of arrays that you wish to process see previous page for details e Mailer Ensure the microarray is placed in the mailer with the printed white s
3. Continued Placing the LifterSlip continued Probing Procedure Protocol continued from previous page 6 LifterSlip nm Array Place one end of the LifterSlip on the array such that the cover slip touches the array just above the nitrocellulose near the barcode Holding the other side with forceps lower the LifterSlip slowly onto the array The dilute serum solution floods the array surface Gently adjust the LifterSlip to remove any air bubbles The LifterSlip is designed to exactly cover the membrane area Cover the chamber Place the chamber containing the array and LifterSlip on a flat surface such that the printed side of the array is facing up and the chamber is as level as possible You can tape the chamber on the flat surface to avoid any accidental disturbances Incubate the array in the chamber for 90 minutes at 4 C without shaking Remove the array from the chamber and insert diagonally into the Array Chamber kept on ice Note The microarray with LifterSlip will not fit on the rails of the chamber You must insert the microarray diagonally into the chamber Using a sterile pipette add 20 ml Probing Buffer page 9 to the chamber wall while keeping the chamber on ice Avoid pipetting buffer directly onto the array surface Gently move the array in the chamber to dislodge the LifterSlip Using forceps carefully remove the LifterSlip without touching the ar
4. and are used as reference spots to orient the microarray and help assign spot identities Human IgG gradient A gradient of human IgG is printed on the microarray The Alexa Fluor 647 goat anti human IgG binds to the human IgG The signals are used to verify the detection reagent and probing procedure Anti Human IgG Ab gradient A gradient of the anti human IgG is printed on the microarray The anti human IgG antibody interacts with the human IgG present in the serum and the interaction is detected by the Alexa Fluor 647 goat anti human IgG antibody The signals are used to verify assay procedure and proper dilution of the serum 23 Troubleshooting Introduction The table below provides some solutions to possible problems you might encounter with the ProtoArray IRBP Application Kit Problem Cause Solution Weak or no Low probe Perform probing with higher serum signal with concentration concentration serum sample Incorrect Follow the recommended protocol for probing probing on page 13 Be sure all incubations are procedure performed at 4 C Prepare the PBST Blocking Buffer and Probing Buffer fresh as described on page 9 Do not allow the array to dry during the probing procedure Avoid prolonged exposure of detection reagents labeled with fluorescent dye to light Incorrect Scan the array at 635 nm and place the array in scanning or the slide holder such that the proteins on the imaging array are facing the laser sou
5. 4 C Module A ProtoArray IRBP 4 C Module B ProtoArray IRBP Room Temperature Room Room Module temperature temperature Proto Array The following components are included in the ProtoArray IRBP 4 C IRBP 4 C Module A Store at 4 C Module A Store Alexa Fluor goat anti human IgG H L at 4 C for up to 3 months For long term storage of up to 6 months aliquot the antibody solution into single use aliquots and store aliquots at 20 C Store the antibody protected from light and avoid repeated freezing and thawing Bovine serum albumin BSA 30 BSA in 0 85 NaCl 240 ml Alexa Fluor 647 goat anti 2 mg ml in 0 1 M sodium 0 5 ml human IgG H L phosphate buffer pH 7 5 0 1 M NaCl 5 mM sodium azide Continued on next page iv Kit Contents and Storage Continued ProtoArray The following components are included in the ProtoArray IRBP 4 C IRBP 4 C Module B Store at 4 C Module B ProtoArray Blocking Buffer 10X 10X PBS pH 7 4 120 ml 1 Tween 20 ProtoArray Probe Buffer 5X 5X PBS pH 7 4 1200 ml 0 25 Triton X 100 25 Glycerol MgCl 1M MgCh in deionized 30 ml water ProtoArray The following components are included in the ProtoArray Room Room Temperature Module Store at room temperature Temperature Module Array Chambers 4 packs of 10 LifterSlip Cover Slips 1 pack of 45 25 mm x 60 mm x 1 mm thick Product Buffers Qualification The ProtoArray Blocking Buffer 10X and Pro
6. Experimental Outline 16 Once you have probed the ProtoArray with the serum sample scan the microarray using a suitable microarray scanner Instructions are included in this section to scan the microarray using a fluorescent microarray scanner You need a suitable scanner to scan the ProtoArray Human Protein Microarray nc To acquire ProtoArray data from the image you need appropriate microarray data acquisition software The recommended microarray data acquisition software for analysis is GenePix Pro Molecular Devices Corporation or ScanArray Software PerkinElmer Inc For scanner specifications see the manual supplied with the array Insert array into the fluorescent microarray scanner Adjust scanner settings 1 2 3 Preview the microarray and adjust settings if needed 4 Scanthe microarray 5 Align grid over spots and use image acquisition software to acquire pixel intensity data 6 Exportand analyze results Continued on next page Scanning Arrays Continued Scanning Procedure A procedure for scanning the ProtoArray Microarrays with a fluorescent microarray scanner is described below For details on using a specific scanner refer to the manual supplied with the scanner The scanning time for each array is 7 8 minutes 1 10 Start the appropriate array acquisition and analysis software on the computer connected to the fluorescence microarray scanner Open the microarray enclosur
7. Microarray nc you probe the ProtoArray Human Protein Microarray nc with the diluted serum sample to detect potential autoantigen biomarkers The ProtoArray detection protocol includes instructions to block the array probe the array with your diluted serum sample wash the array to minimize non specific interactions detect interactions using the Alexa Fluor 647 anti human IgG Antibody wash to remove unbound antibody dry scan the array to acquire high resolution array image view results and analyze data For a detailed experimental workflow see page 5 Using the ProtoArray IRBP Application Kit to perform immune response biomarker profiling offers the following advantages e Provides a simple efficient and rapid method to screen for interactions of serum antibodies against thousands of human proteins within a day e Includes qualified buffers and detection reagents for probing eliminating the need to prepare reagents e Provides sensitive stable fluorescence detection using the Alexa Fluor 647 dye The ProtoArray IRBP 4 C Modules A and B include qualified reagents used in the blocking washing and detection steps during probing of the ProtoArray Microarrays The pre made buffers provide consistent results and eliminate the time required to prepare reagents Continued on next page Overview Continued Alexa Fluor 647 Detection ProtoArray IRBP Room Temperature Module ProtoArray Central Portal The h
8. be Buffer 5X are diluted to 1X with deionized water and subjected to pH and conductivity measurements The pH and conductivity for each buffer must be within the specified range Alexa Fluor 647 goat anti human IgG The spectra for Alexa Fluor 647 goat anti human IgG antibody must indicate absorption maxima of 650 nm and emission maxima of 668 nm The degree of labeling is verified and must contain 2 8 fluorophore per IgG molecule Accessory Products Additional The table below lists additional products available Products separately from Invitrogen For more information about these products visit www invitrogen com or call Technical Support page 26 Product Quantity Catalog no ProtoArray Human Protein Microarray nc v4 0 1 array PAH052401 ProtoArray Human Protein Microarrays nc 20 arrays PAHO0524015 v4 0 Pilot Study Bundle ProtoArray Human Protein Microarrays nc 40 arrays PAHO0524017 v4 0 Discovery Study Bundle A ProtoArray Human Protein Microarrays nc 80 arrays PAHO0524019 v4 0 Discovery Study Bundle B vi Overview Introduction Kit Components Introduction Serum profiling is an important method to identify differentially expressed antibodies in healthy and diseased samples Using the ProtoArray Technology for serum profiling has been demonstrated to be a rapid and sensitive method to detect potential autoantigen biomarkers Mattoon et al 2005 Michaud et al 2003 The ProtoAr
9. clusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Limited Use Label License No 5 Invitrogen Technology The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagno
10. d for different applications For example ProtoArray Prospector uses the file HA10756 IRP PAI to analyze data from IRBP experiments performed on array from lot HA10756 Continued on next page 19 Data Acquisition and Analysis Continued ProtoArray Central continued 20 Slide Information File LotNumber slide txt This file contains a listing of all barcodes associated with a specific lot of arrays Download the files listed above for human array specific information from a specific lot Use these files to interpret your results with the ProtoArray Human Microarray nc Note The file size for some files such as the Protein Sequence File may be larger than 1 MB To acquire data from ProtoArray experiments e ForGenePix Pro Software download the GAL files from ProtoArray Central for protein arrays which define the array grid required by the microarray data acquisition software e For other microarray data acquisition software use data from the GAL files from ProtoArray Central for protein arrays to generate files that are compatible with your microarray data acquisition software to define the array grid Scroll through the image to ensure that the grid is in proper location for each subarray Adjust the subarray grid if needed After the grid is properly adjusted and all of the features are aligned acquire the pixel intensity data Save export the results as a GPR GenePix Results file for data a
11. de S Gu Y Lian W Megginson M She J X and Wu D 2002 Protein Microarrays to Detect Protein Protein Interactions Using Red and Green Fluorescent Proteins Anal Biochem 306 50 54 Mattoon D Michaud G Merkel J and Schweitzer B 2005 Biomarker Discovery Using Protein Microarray Technology Platforms Antibody antigen Complex Profiling Expert Rev Proteomics 2 879 889 Michaud G A Salcius M Zhou F Bangham R Bonin J Guo H Snyder M Predki P and Schweitzer B 2003 Analyzing Antibody Specificity With Whole Proteome Microarrays Nature Biotechnol 21 1509 1512 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use Trademarks referenced herein are either registered trademarks or trademarks of Invitrogen Corporation Any registration or trademark symbols used herein denote the registration status of trademarks in the United States Trademarks may or may not be registered in other countries LifterSlip is a trademark of Erie Scientific Company 28 invitrogen Corporate Headquarters Invi Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country ific contact information visit our web site at www invitrogen com
12. e chamber on ice Avoid pipetting buffer directly onto the array surface Incubate the array in Probing Buffer for 8 minutes at 4 C with gentle shaking Decant the buffer Drain excess buffer by inverting chamber on paper towels for a few seconds Repeat Steps 12 13 two more times using 20 ml Probing Buffer each time to perform a total of 3 washing steps Proceed to Drying the Array next page Continued on next page Performing Serum Profiling Continued Drying the Array Remove the array from the chamber at the end of the probing procedure Tap one edge of the array gently on a laboratory wipe for a few seconds to drain any buffer Place the array in a slide holder or a sterile 50 ml conical tube if you do not have a slide holder in a vertical orientation Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Centrifuge the array in the slide holder or 50 ml conical tube at 800 x g for 3 5 minutes in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Place the array in a slide box and keep the box with the lid open in the dark for 30 60 minutes at room temperature to dry the array Make sure that the array is completely dry there should be no translucent areas Scan the array using a fluorescence microarray scanner see next page for details 15 Scanning Arrays Introduction Materials Needed
13. e on the scanner Place the ProtoArray Human Protein Microarray nc in the holder such that the nitrocellulose coated side of the array faces the laser source and barcode on the array is closest to the outside of the instrument Close the microarray enclosure on the scanner Adjust the settings to image the microarray using the recommended settings for GenePix scanner Molecular Devices Corporation listed below e Wavelength 635 nm e PMT Gain 600 e Laser Power 100 e Pixel Size 10 um e Lines to Average 1 0 e Focus Position 0 um Perform a preview to quickly scan the microarray Adjust the PMT Gain if needed Human IgG is printed in at least 2 concentrations on each subarray To optimize the dynamic range of observed signals set the PMT gain such that the highest concentration of human IgG exhibits a signal just above saturation Note The image should have very few saturated spots white Select the area of the array to scan in detail include the barcode in the area for record and then scan the array to provide a high resolution image After acquiring the image save the image to a suitable location as multi image TIFF file Be sure the barcode is included in the name of the image Open the microarray enclosure and remove the microarray from the holder Proceed to downloading lot specific information next page 17 Data Acquisition and Analysis Introduction GAL File Important ProtoArray Cent
14. h serum samples Prior to use process the serum sample to remove any aggregates by centrifugation 12000 x g for 30 seconds on a microcentrifuge if needed We recommend using a 1 150 dilution of the serum sample in Probing Buffer to maximize signals while minimizing false positive and false negative results Based on your initial results you may need to optimize the serum dilution to obtain optimal performance Continued on next page 7 Performing Serum Profiling Continued Materials Needed Experimental Outline Po Human serum sample dilute the serum sample 1 150 in Probing Buffer store on ice until use ProtoArray Human Protein Microarray nc v4 0 page vi ProtoArray IRBP 4 C Module A supplied with the kit ProtoArray IRBP 4 C Module B supplied with the kit ProtoArray IRBP Room Temperature Module supplied with the kit Optional Glass slide chamber VWR catalog no 74280 014 or equivalent Sterile covered chamber for incubation you may use a 50 ml conical tube petri dish or plastic container with lid which is sterilized with bleach Forceps Deionized water Block the ProtoArray Human Protein Microarray nc Probe the array with diluted human serum Perform detection with the Alexa Fluor 647 goat anti human IgG Dry the array for scanning Scan the array to obtain an array image Acquire the image data using a microarray data acquisition software Analyze results using ProtoArray P
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16. ide facing up Add 30 ml PBST Blocking Buffer recipe is on page 9 to the mailer containing the human microarray Incubate for 1 hour at 4C with gentle shaking 50 rpm e Glass Slide Chamber Add 120 ml PBST Blocking Buffer recipe on page 9 to the glass slide chamber Insert the microarrays in the chamber there are 10 slots in the glass chamber but the slots are wide enough to accommodate 20 arrays arranged back to back Incubate for 1 hour at 4 C with gentle shaking 750 rpm Remove arrays from the blocking buffer as follows e Removal from Mailer Decant the PBST Blocking Buffer Drain excess buffer by inverting the mailer on paper towels for a few seconds Remove the array from the mailer Tap one edge of the array gently on a laboratory wipe for a few seconds to drain any buffer without allowing the array to dry e Removal from Glass Slide Chamber Remove each slide one at a time from the chamber and gently tap one edge of the array on a laboratory wipe for a few seconds to drain any buffer without allowing the array to dry Proceed immediately to Placing the LifterSlip next page Continued on next page 11 Performing Serum Profiling Continued Placing the LifterSlip 12 1 Immediately remove a LifterSlip from the package and place the LifterSlip on a dark surface with the raised edges facing up Note The LifterSlip has raised edges white in color on the slip on each of the long sides
17. igh sensitivity low background signal stability and commercial availability of fluorescence microarray scanners make fluorescence detection the preferred method for detecting protein protein interactions on microarrays The ProtoArray IRBP Application Kit includes the Alexa Fluor 647 goat anti human IgG H L Antibody for detection of the serum immunoglobulin The Alexa Fluor 647 fluorophore is brighter and more stable than other commercially available dyes such as Cy Dyes and is more sensitive for detecting interactions on protein arrays We have demonstrated that detection with Alexa Fluor 647 produces approximately 2 fold higher signal background ratios than Cy5 detection ProtoArray IRBP Room Temperature Module includes Array Chambers required for use during washing steps and LifterSlip cover slips LifterSlip cover slips included with the kit are 1 mm thick 25 x 60 mm glass cover slips with a raised edge design that allows for even dispersal of solutions between the array and coverslip The cover slips hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents See page 6 for details on LifterSlip and page 12 for using the LifterSlip The ProtoArray Central Portal at www invitrogen com protoarray provides a web based user interface to access ProtoArray specific information including online tools applications and other resources You also use the por
18. in Microarray nc v4 0 The ProtoArray Human Protein Microarrays are high density protein microarrays containing thousands of human proteins Each human open reading frame ORF is expressed as an N terminal GST fusion protein purified and printed in duplicate on a nitrocellulose coated glass slide The use of nitrocellulose as a surface to print the arrays ensures maximum utility for protein assays since the nitrocellulose surface is known to be compatible with a variety of protein functions Espejo et al 2002 Hesselberth et al 2006 Kukar et al 2002 The nitrocellulose coating is thin and does not interfere with scanning of the array The ProtoArray Human Protein Microarray nc v4 0 is specifically designed with suitable controls printed on the array to allow serum profiling using serum For array specifications and details on the human microarray refer to the manual supplied with the array LifterSlip cover slips included with the kit are 1 mm thick glass cover slips with a raised edge design that allows for even dispersal of solutions between the array and coverslip This provides increased data quality by eliminating gradients caused by floating standard coverslips and minimizes non uniform labeling Capillary attraction ensures that LifterSlips stay in position The increased stiffness and superior surface quality of the LifterSlip ensures consistent dispersal of solutions without any flexing or bowing of the covers
19. invitrogen ProtoArray Immune Response Biomarker Profiling Application Kit For serum profiling applications Catalog no PA016 Version A 13 September 2006 25 0971 ii Table of Contents Table of Contents ire eet ble ee E EE r iii Kit Contents and Storage ienris iiser en Eisi een eene iv Accessory Prod cts c teneret eter t scandere ide vi Introduction nain conor nna a arn Ra Cro Rao ra Sra Exe ra xu san saw ooa p arua Darin 1 QVervieW isestetiatein teet regie e E erede e Tree Ee e 1 Experimental Overview teeitee ie ree rette reign 5 lien mE 6 Performing Serum Profiling sssssseeeeeneeeeennen nene 6 Scanning Arrays Data Acquisition and Analysis sse enne 18 Expected Results tte cem ete tt escam tentent 22 Troubleshooting chier eet reete ttis 24 DNgcn pem ealanta haii 26 Technical Support een ettet ere nett Ee eds 26 Purchaser Notification Referetices ie eec diii e i A b a UP etd 28 iii Kit Contents and Storage Shipping and TheProtoArray Immune Response Biomarker Profiling Storage IRBP Kit consists of 3 modules and is shipped as detailed below Upon receipt store each module as indicated For details on each component see below Sufficient reagents are included to perform 40 microarray screening experiments All kit components are stable for 6 months when stored properly ProtoArray IRBP
20. lip Continued on next page Performing Serum Profiling Continued Important RECO Nos Serum Sample Use the probing procedure from this section as a starting protocol and based on your initial results optimize the probing protocol by varying the serum concentrations To obtain the best results with ProtoArray follow these guidelines e Always wear clean gloves while handling microarrays e Do not touch the surface of the array to avoid any damage to the array surface resulting in uneven or high background e Maintain the array and reagents at 2 8 C during the experiment e Avoid drying of the array during the experiment and ensure the array is completely covered with the appropriate reagent during all steps of the protocol e Be sure to take the appropriate precautions wear a laboratory coat disposable gloves and eye protection when handling serum samples and dispose of serum samples as biohazardous waste e Process the serum samples centrifuge to remove any aggregates prior to application on the array if needed e Use ProtoArray Human Protein Microarray nc v4 0 for serum profiling applications Do not use any other array The serum profiling application has been optimized for use with human serum samples fresh or frozen For information on using the ProtoArray Human Protein Microarray with other biological fluids contact Technical Support page 26 Avoid repeated freeze thaw cycles wit
21. media as described below and then download the information onto the scanner computer 1 Connectto the portal at www invitrogen com protoarray Click on the Online Tools tab 2 AProtoArray Lot Specific Information page is shown Continued on next page Data Acquisition and Analysis Continued ProtoArray Central continued Procedure continued from previous page Enter the array barcode in the Input Barcode Number box Click on the Search button invitrog For each input barcode the following files are displayed GAL file LotNumber gal This file is essential for data acquisition by the software and defines spot locations and identities of all protein spots on the array The file also includes the equivalent solution protein concentration in nM for use during data analysis Protein Information File LotNumber_info txt This file contains a listing and description of the human proteins on the microarray Protein Sequence File LotNumber seq txt This tab delimited text file lists the GenBank accession number Ultimate ORF Clone ID number if available FASTA header and amino acid sequence of the ORF for each array protein Control Data File LotNumber control txt This file contains a description of control spots on the array Protein Application File LotNumber_application PAD ProtoArray Prospector uses the Protein Application Files for data analysis Different PAI files are designe
22. nalysis using ProtoArray Prospector see next page The results contain the pixel intensity information for each spot feature on the array and information on additional parameters depending on the type of software used for data acquisition Alternatively save export the results with an xls extension or rename the tab or gpr file using the xls extension for data analysis using Microsoft Excel Continued on next page Data Acquisition and Analysis Continued Data Analysis Using ProtoArray Prospector After data acquisition analyze the data to identify potential biomarkers Visual identification of interactions can be performed after initial identification of significant interactions is done using the data analysis guidelines listed below We recommend using the ProtoArray Prospector software available from Invitrogen for data analysis The ProtoArray Prospector software quickly analyzes the data generated from the image acquisition software and easily identifies statistically significant interactors saving you time and effort without the need to perform any manual calculations In addition the software has features that allow you to modify the analysis method and compare data obtained from different arrays The ProtoArray Prospector software and manual are available for FREE to ProtoArray users To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click on the Onli
23. ne Tools tab Install the Complete version of ProtoArray Prospector which additionally installs the Serum Profiling Toolbox that includes a more comprehensive set of algorithms for the Immune Response Profiling application The ProtoArray Prospector software currently accepts as input files the output files GPR generated by the GenePix Pro microarray data acquisition software and ProtoArray Prospector analyzes the data using specified algorithms to generate a list of human proteins showing significant interactions with the serum sample If GPR files are not available consult the ProtoArray Prospector manual for guidelines to format a results file that is compatible for import into ProtoArray Prospector 21 Expected Results Introduction Results obtained after probing the ProtoArray Human Protein Microarray nc v4 0 with human serum are shown below Probed with Alexa Fluor 647 goat anti human IgG antibody only Probed with 1 150 diluted human serum and Alexa Fluor 647 goat anti human IgG antibody Human Array Image Boxed area shown in detail Human Array Boxed area shown Image in detail Alexa Fluor Ab Alexa Fluor Ab Human IgG 22 Continued on next page Expected Results Continued Results continued Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array is properly scanned
24. p to the array as described properly on page 12 To avoid drying of the membrane make sure the cover slip covers the membrane area of the array and adjust the cover slip if needed Serum or Centrifuge the serum sample or detection detection reagents to remove precipitates prior to reagents probing the array contain precipitates LifterSlip Be sure to use LifterSlip cover slips included cover slips not with the kit during probing The LifterSlip used cover slips are designed with a raised edges that allow for even dispersal of solutions between the array and coverslip Do not use HybriSlip or any other glass cover slips 25 Appendix Technical Support Contact Us For more information or technical assistance please call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 444 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail tech supportGinvitrogen com E mail eurotech invitrogen com MSDS Limited Warranty 26 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with
25. ral 18 After scanning and saving an image of the array download the protein array lot specific information including the GAL file from the ProtoArray Central Portal You will use the lot specific information to acquire and analyze the data to identify antibody protein interactions The GAL GenePix Array List files describe the location and identity of all spots on the protein microarray and are used with the microarray data acquisition software to generate output files that contain pixel intensity information for all features on the slide The GAL files are available for downloading from the ProtoArray Lot Specific Information available on ProtoArray Central see below While downloading the lot specific information files ensure that you are downloading files that are associated with your specific barcode on the array Since lot specific information files are updated frequently based on recently available sequence or protein information make sure that you download the latest version of the lot specific information files The ProtoArray Central Portal provides a web based user interface to retrieve ProtoArray Lot Specific information This information GAL file is required for acquiring the array data If the scanner computer is connected to the internet then click on the link below to connect to the portal If the scanner computer is not connected to the internet download the array specific information to portable
26. ray Immune Response Biomarker Profiling IRBP Application Kit when used with ProtoArray Human Protein Microarray nc nitrocellulose v4 0 provides a rapid efficient and sensitive method to screen for interactions of serum antibodies of interest against thousands of human proteins printed on the array The screening is complete within a day as compared to other methods such as western blotting The ProtoArray Human Protein Microarray nc v4 0 contains thousands of human proteins expressed as N terminal glutathione S transferase GST fusion proteins purified and printed in duplicate on a nitrocellulose coated glass slide The ProtoArray Human Protein Microarray nc v4 0 is specifically designed with suitable controls for serum profiling application The major components of the ProtoArray IRBP Application Kit are e The ProtoArray IRBP Buffer Modules A and B contain pre made qualified reagents for performing the blocking and washing steps during probing e The Alexa Fluor 647 goat anti human IgG Antibody for detection e The ProtoArray IRBP Room Temperature Module contains the Array Chambers and LifterSlip glass cover slips used for incubation and washing steps For more information on each component see next page Continued on next page Overview Continued System Overview Advantages ProtoArray IRBP 4 C Modules A and B To use the ProtoArray IRBP Application Kit with the ProtoArray Human Protein
27. ray surface Discard the LifterSlip Reposition the array on the bottom chamber rails such that maximal coverage of solution occurs above the array Continued on next page 13 Performing Serum Profiling Continued Probing Procedure continued from previous page Procedure 5 continued 10 11 12 13 14 15 14 Incubate the array in Probing Buffer for 8 minutes at 4 C with gentle shaking Decant the Probing Buffer Invert chamber on paper towels for a few seconds to drain excess buffer Using a sterile pipette add 20 ml Probing Buffer page 9 to the chamber wall while keeping the chamber on ice Avoid pipetting buffer directly onto the array surface Incubate the array in Probing Buffer for 8 minutes at 4 C with gentle shaking Decant the Probing Buffer Invert chamber on paper towels for a few seconds to drain excess buffer While the array is incubating mix 12 5 ul Alexa Fluor 647 goat anti human IgG antibody with 25 ml Probing Buffer to obtain a final antibody concentration of 1 ug ml Repeat Steps 6 7 once more using 20 ml Probing Buffer to obtain a total of 3 wash steps Add 25 ml Alexa Fluor 647 Antibody solution from Step 8 to the chamber Incubate the chamber for 90 minutes at 4 C with gentle shaking in the dark Decant the buffer Invert the chamber on paper towels for a few seconds to drain excess buffer Slowly add 20 ml Probing Buffer onto the chamber wall while keeping th
28. rce High Improper Prepare the PBST Blocking Buffer fresh as background blocking described on page 9 Improper To obtain the best results perform the washing recommended washing steps Prepare the Probing Buffer fresh as described on page 9 Array dried Do not allow the array to dry during probing during probing Array not dried Dry the array as described before scanning properly before scanning High probe Decrease the serum concentration concentration Continued on next page 24 Troubleshooting Continued High Particulate Be sure to remove any particulate material background material in from serum samples using centrifugation serum sample Uneven Uneven During the blocking or washing steps ensure background blocking or the array is completely immersed in blocking washing solution or Probing Buffer and use at least 25 ml buffer in the Array Chamber to cover the array completely with buffer Improper To obtain the best results perform the washing recommended washing steps Prepare the Probing Buffer fresh as described on page 9 Portions of Do not allow the array to dry during probing array have dried Improper array Always wear gloves and avoid touching the handling surface of the array with gloved hands or forceps Take care while inserting the array into the Array Chamber to avoid scratching the array surface Serum sample Apply the diluted serum solution and not applied LifterSlip cover sli
29. rospector Continued on next page Performing Serum Profiling Continued Preparing PBST Blocking Buffer Preparing Probing Buffer Prepare the PBST Blocking Buffer fresh prior to use The recipe below provides sufficient buffer to probe 1 2 microarrays in a mailer 30 ml or up to 20 microarrays in glass slide chambers 120 ml PBST Blocking Buffer 1X PBS 1 BSA 0 1 Tween 20 1 Prepare PBST Blocking Buffer using reagents from the ProtoArray IRBP Application Kit as follows Reagents 30 ml vol 120 ml vol Blocking Buffer 10X 3ml 12 ml 30 BSA 1ml 4ml Deionized water to 30 ml to 120 ml 2 Mix well do not vortex and store on ice until use After preparing the Blocking Buffer immediately return the remaining 30 BSA to 20 C Prepare the following buffer fresh prior to use The recipe below provides sufficient buffer to probe one microarray Probing Buffer 1X PBS 5 mM MgCb 0 05 Triton X 100 5 Glycerol 1 BSA 1 Prepare 150 ml Probing Buffer using reagents from the ProtoArray IRBP Application Kit as follows Probe Buffer 5X 30 ml 1M MgCb 750 ul 30 BSA 5 ml Deionized water to 150 ml 2 Mix well do not vortex and store on ice until use Continued on next page Performing Serum Profiling Continued Before Starting Note 10 Before starting the probing procedure make sure you have all items on hand especially buffers previous page serum diluted in Probing Buffer page 7
30. stic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 27 References Espejo A Cote J Bednarek A Richard S and Bedford M T 2002 A Protein Domain Microarray Identifies Novel Protein Protein Interactions Biochem J 367 697 702 Hesselberth J R P M J A G J E S Michaud G A and S F 2006 Comparative Analysis of Saccharomyces cerevisiae WW Domains and their Interacting Proteins Genome Biol 7 R30 Kukar T Eckenro
31. tal to retrieve ProtoArray Lot Specific information page 18 which is required for analysis of the array data and identification of statistically significant interactions Continued on next page Overview Continued ProtoArray Prospector The ProtoArray Prospector software quickly analyzes the microarray data generated by the data acquisition software and easily identifies significant hits saving you time and effort In addition the software has features that allow you to modify the analysis method and compare data obtained from different microarrays The ProtoArray Prospector software and manual are available for FREE to ProtoArray users To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click on Online Tools tab Experimental Overview Experimental The experimental outline for immune response biomarker Outline profiling is described below Dilute Serum Sample Probe Human Array with Diluted Serum Sample Scan Array and Acquire Array Image Analyze Results Using ProtoArray Prospector Methods Performing Serum Profiling Introduction ProtoArray Human Protein Microarray LifterSlip Cover Slips To use the ProtoArray IRBP Application Kit you will need to purchase ProtoArray Human Protein Microarray nc v4 0 available from Invitrogen page vi Use the protocol described in this section to perform serum profiling using a ProtoArray Human Prote
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