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User Manual - Thermo Fisher Scientific

1. Product Qualification Introduction ProtoArray Human Yeast and Control Microarrays Array Control Protein ProtoArray PPI Buffers Module ProtoArray Mini Biotinylation Module ProtoArray Biotinylation Purification Module The components supplied in the ProtoArray Human Protein and Yeast Proteome Microarray PPI Complete Kits are qualified as described below The ProtoArray Human Protein Yeast Proteome and Control Microarrays are visually examined for obvious defects The quality of the printing process is verified by probing several arrays from each lot with an anti GST antibody The scanned image of the array must show a uniform spotting pattern The arrays are also functionally qualified by probing with the Array Control Protein biotinylated calmodulin kinase probe to ensure that appropriate interactions and controls are detected The Array Control Protein biotinylated calmodulin kinase is qualified by performing a Western detection with streptavidin AP and must show that the protein is biotinylated The protein concentration of the Array Control Protein must be within the specified range The 10X Blocking Buffer and 5X Probe Buffer 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 Biotin XX Sulfosuccinimidyl Ester The Biotin XX sulfosuccinimidyl ester must be gt 90 p
2. 53 Appendix Technical Support Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech _supportGinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty 54 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you
3. Application Kit you also need to purchase a ProtoArray Human Yeast or Control Protein Microarray nc e 2ProtoArray Control Protein Microarrays nc v4 0 included in the complete kits only or available separately e ProtoArray PPI Buffer Modules A and B included with the kit e Streptavidin Alexa Fluor 647 Conjugate included with the kit keep on ice in the dark until immediately before use e Biotinylated Protein Probe in Probing Buffer see next page e Array Control Protein in Probing Buffer included in the complete kits only see next page e 2sterile 50 ml conical tubes e Ice bucket e Deionized water e Optional Microarray slide holder and centrifuge equipped with a plate holder Each ProtoArray Control Protein Microarray can only be used once Do not re use the microarray or reprobe the same microarray with another probe Continued on next page Probing the ProtoArray Control Protein Microarrays Continued Experimental 1 Block the ProtoArray Control Protein Microarrays Outline 2 Probe one array with the biotinylated protein probe and the other with the y y P P Array Control Protein 3 Perform detection with the Streptavidin Alexa Fluor 647 Conjugate Dry the arrays for scanning Important Since proteins are sensitive to various environmental factors each array is Guidelines produced in an environment controlled facility to ensure protein integrity and maintain consistency To obt
4. Central portal see page 42 The ProtoArray Control Protein Microarray specifications are listed below The proteins on the microarray are printed in 48 subarrays and are equally spaced in vertical and horizontal directions For details on the subarray layout and yeast protein and control spots on the ProtoArray Control Protein Microarray nc go to the ProtoArray Central Portal at www invitrogen com protoarray Total Subarrays 48 4 columns x 12 rows Subarray Size 4400 um x 4400 um Subarray Dimensions 8 rows x 20 columns Median Spot Diameter 150 um Spot Center to Center Spacing 220 um Distance Between Subarrays 100 um Replicates per Sample 2 Continued on next page 11 ProtoArray Control Protein Microarray Continued Controls Printed on Each ProtoArray Microarray Various proteins and controls are printed on each ProtoArray Human Protein Yeast Proteome and Control Protein Microarray nc to allow you to verify reagents background and detection conditions used during probing The table below lists the controls printed on each ProtoArray Microarray Protein Function Control Spots required for PPI Data Analysis Alexa Fluor Antibody Rabbit anti mouse IgG Antibody labeled with Alexa Fluor 647 Alexa Fluor 555 and Alexa Fluor 488 Serves as a positive control for fluorescence scanning and for orientation of the microarray image Bovine Serum Albumin
5. 647 Detection ProtoArray PPI Application Kit ProtoArray Central Portal ProtoArray Prospector The ProtoArray PPI Buffer Module 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 ProtoArray PPI Buffers Module B includes HybriSlip cover slips that hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents Array Chambers are also included in the module for washing the microarrays The high 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 Human Protein and Yeast Proteome Microarray PPI Kits include the Streptavidin Alexa Fluor 647 Conjugate for detection of the biotinylated protein probe 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 The ProtoArray Protein Protein Interaction Application Kit includes ProtoArray
6. Molecular Weight Standard Gel 2 Lane Sample Final Amount in Gel 1 Biotinylated Standard 200 fmoles biotin 2 Biotinylated Standard 100 fmoles biotin 3 Biotinylated Standard 50 fmoles biotin 4 Biotinylated Standard 25 fmoles biotin 5 Biotinylated Standard 12 5 fmoles biotin 6 Biotinylated protein 3 1 100 fmoles protein 7 Biotinylated protein 9 1 100 fmoles protein 8 Biotinylated protein 27 1 100 fmoles protein 9 Biotinylated BSA 9 1 100 fmoles protein 10 Molecular Weight Standard a For NuPAGE Novex Bis Tris Gels perform SDS PAGE at 200 V for 35 50 minutes using NuPAGE MES or MOPS Running Buffer with an XCell SureLock Mini Cell Remember to include NuPAGE Antioxidant in the running buffer see instructions included with the NuPAGE gels After electrophoresis is complete proceed to blotting next page Continued on next page Assessing Protein Biotinylation Continued Protein Transfer General Guidelines Preparing Solutions for Nitrocellulose Membranes Transfer proteins from the two gels to nitrocellulose membranes using a suitable transfer apparatus Note PVDF membranes are not recommended page 23 for use in Western transfer when using this protocol For NuPAGE Novex Bis Tris Gels perform transfer at 30 V for 1 hour using 1X NuPAGE Transfer Buffer with 10 methanol After transfer proceed to detection and visualization as described below To obtain
7. BSA A negative control for non specific protein interactions Biotinylated Anti mouse A positive control for interaction with streptavidin labeled Antibody detection reagent Anti biotin Antibody Detects biotinylated probes V5 Control Protein biotinylated V5 tagged control protein A positive control for detection with the Anti V5 Alexa Fluor 647 Antibody Human IgG Protein Gradient A positive control for the immune response serum profiling application Interacts with Alexa Fluor 647 goat anti human IgG Anti Human IgG Antibody Gradient goat anti human IgG A positive control for the immune response serum profiling application Interacts with serum IgG antibodies which are then bound by Alexa Fluor 647 goat anti human IgG Yeast calmodulin Cmd1p A positive control for protein protein interaction application and interacts with the Array Control Protein GST Protein Gradient Serves as a negative control and signals are used by ProtoArray Prospector software for background and statistical significance calculations Control Spots NOT required for PPI Data Analysis Fiduciary Kinases Kinases autophosphorylate and produce fiduciary marker signals which are used for orientation of the microarray image also serves as a positive control for the radiolabel and assay conditions Control Kinase Substrate A substrate for the Control Kinase used to verify assay conditions The Control K
8. Biotinylation Purification Module a ee ee a a BEN AS a a a a A A pues ProtoArray Biotinylation Assessment Module Continued on next page Kit Contents and Storage Continued Shipping and Storage ProtoArray Human or Yeast Microarrays ProtoArray Control Reagents vi The components included in the ProtoArray PPI Kits for Biotinylated Proteins are shipped as detailed below Upon receipt store as indicated All kit components are stable for 12 months when stored properly Shipping ProtoArray Human Protein Microarray nc v4 0 Blue ice ProtoArray Yeast Proteome Microarray nc v1 1 Blue ice ProtoArray Control Protein Microarray nc v4 0 Blue ice Array Control Protein Dry ice Streptavidin Alexa Fluor 647 Conjugate Blue ice ProtoArray PPI Buffer Module A Dry ice ProtoArray PPI Buffer Module B Blue ice ProtoArray Mini Biotinylation Module Dry ice ProtoArray Biotinylation Purification Module Blue ice ProtoArray Biotinylation Assessment Module Blue ice Each ProtoArray Microarray PPI Complete Kit contains mailers with the following ProtoArray Microarrays e Human Kit Catalog no PAH0524011 Contains two ProtoArray Human Protein Microarrays nc v4 0 e Yeast Kit Catalog no PA0121011 Contains two ProtoArray Yeast Proteome Microarrays nc v1 1 Store the microarrays at 20 C For details on array specifications see pages 6 11 Each ProtoArray Microarray PPI Complete
9. Excitation 635 nm or equivalent Detection limit 0 1 fluor um Resolution lt 10 um Dynamic Range gt 3 orders of magnitude Output 16 bit TIFF 39 Scanning Arrays Continued Recommended Scanners Note 40 The following scanners are compatible for scanning ProtoArray Human Protein or Yeast Proteome Microarrays e GenePix 4000A Molecular Devices Corporation GenePix 4000B Molecular Devices Corporation e GenePix Professional 4200A Molecular Devices Corporation e GenePix Personal 4100A Molecular Devices Corporation e ScanArray Lite PerkinElmer Inc e ScanArray Express PerkinElmer Inc e ScanArray Express HT PerkinElmer Inc e LS Series Laser Scanner Tecan Group AG The following scanners may be compatible with ProtoArray Human Protein or Yeast Proteome Microarrays e AlphaArray Reader Alpha Innotech Corporation e arrayWoRx 4 Color Biochip Reader Applied Precision LLC e SpotLight TeleChem International Inc The following scanners are not compatible with ProtoArray Human or Yeast Microarrays e GeneChip Scanner 3000 Affymetrix Inc e DNA Microarray Scanner Agilent Technologies Inc Unlike most DNA microarrays you will scan the ProtoArray Human Protein or Yeast Proteome Microarray nc using only one color Continued on next page Scanning Arrays Continued Scanning Procedure Note A brief procedure for scanning th
10. Kit includes the following control reagents Store the microarray and Array Control Protein at 20 C ProtoArray Control Protein 2 arrays Microarray nc v4 0 Array Control Protein 0 5 mg ml in phosphate 40 ul biotinylated calmodulin kinase buffered saline PBS pH 7 4 with a V5 tag For details on array specifications see page 11 For information about the Array Control Protein see page 32 Continued on next page Kit Contents and Storage Continued Streptavidin Alexa The ProtoArray Microarray PPI Complete Kits and ProtoArray Protein Fluor 647 Protein Interaction Application Kit each contain 1 tube of Streptavidin Alexa Conjugate Fluor 647 Conjugate with the following specifications e Concentration 2 mg ml in phosphate buffered saline PBS pH 7 2 with 5 mM sodium azide e Amount supplied 30 ul Store at 4 C Protect the Streptavidin Alexa Fluor 647 Conjugate from exposure to light ProtoArray PPI The ProtoArray PPI Buffer Module A includes the following reagents Buffer Module A Store at 20 C Note The amount of reagents supplied is sufficient to perform 4 microarray screening experiments Bovine Serum Albumin BSA 30 BSA in 0 85 NaCl 30 ml DTT 1 M DTT in deionized water 400 ul ProtoArray PPI The ProtoArray PPI Buffer Module B includes the following reagents Note The amount of reagents supplied is sufficient to perform 4 microarray screening experiments ProtoAr
11. PBST Blocking Buffer and Probing Buffer as described on page 32 You need 120 ul of your biotinylated protein probe Use the biotinylated probe that gives the best signal on the Western blot at the lowest biotinylation molar ratio Dilute the probe to 5 50 ug ml in Probing Buffer Mix well do not vortex and store on ice until use e Before starting the probing procedure make sure you have all items on hand especially buffers see previous page probes in Probing Buffer see previous page Array Chambers included in the kit and HybriSlips included in the kit e Make sure the buffers are cold Store buffers on ice until use Place the Array Chamber on ice to chill the chamber until use M e Review Important Guidelines on page 31 prior to starting the probing procedure Continued on next page 37 Probing the ProtoArray Human or Yeast Microarrays Continued Probing Arrays 38 The options for probing the array are described on the previous page Choose the option that best fits your needs 1 Probe the ProtoArray Human Protein or Yeast Proteome Microarray nc using the procedure described on page 33 2 Dry the array as described on page 35 3 Scan the arrays as described on the next page and analyze results page 42 Examples of expected results obtained after probing the ProtoArray Human Protein or Yeast Proteome Microarrays are shown on pages 47 and 48 respectively If you obtain weak sig
12. centrifuge briefly in a microcentrifuge at maximum speed Check the pH of the solution as incorrect pH may result in failed biotinylation Spot a small amount of the mixture 1 ul from Step 1 ona 7 10 pH paper and compare color with sodium bicarbonate buffer The pH of the solution should be 8 0 If the pH is not 8 0 discard samples and dialyze purified protein against PBS Prepare samples as described in Step 1 with dialyzed samples A formula is included below to calculate the amount of biotin reagent required for protein biotinylation in Step 6 next page If you are an experienced user and are familiar with protein molar calculations you may use your own calculation Use the formula below to calculate the amount of biotin reagent to use 90 000 pl biotin reagent to be added to protein samples MW Da MW is the molecular weight of the protein probe in Daltons Example The molecular weight of your protein sample is 50 000 Da Calculate the amount of biotin reagent required as follows 90 000 1 8 ul biotin reagent to be added to protein samples 50 000 Continued on next page 19 In Vitro Biotinylation Continued Important Biotinylation Reaction 20 The reactive form of biotin XX sulfosuccinimidy l ester rapidly hydrolyzes in water Prior to dissolving biotin XX sulfosuccinimidy ester in water Label and set up the dilution tubes as described below on ice e Calculate the amount of biotin reagent requir
13. columns Median Spot Diameter 150 um Spot Center to Center Spacing 220 um Distance Between Subarrays 100 um Replicates per Sample 2 Total Human Proteins on v4 0 array 8000 Refer to ProtoArray Central Portal for exact number of human proteins printed on the microarray Continued on next page ProtoArray Human Protein Microarray Continued TM Array Content The majority of human protein collection is derived from the human Ultimate ORF open reading frame Clone Collection available from Invitrogen see http orf invitrogen com for more information Each Ultimate ORF Clone is full insert sequenced and is guaranteed to match the corresponding GenBank amino acid sequence Some of the human proteins printed on the array represent the human protein kinase collection derived from full insert sequenced clones but are not Ultimate ORF Clones Some of the kinases from the kinase collection have been cloned as catalytic domains rather than full length proteins About 250 proteins printed on the array are derived from the purified protein kinase collection available from Invitrogen Approximately 25 proteins peptides and nucleic acids that have been demonstrated to be antigens in a variety of autoimmune diseases are also printed on the array For accession number and amino acid sequence for each protein as well as information on peptides and nucleic acids printed on the array download the Protein Information F
14. conjugate must indicate an Fluor 647 absorption maxima 654 5 nm and emission maxima of 669 5 nm The degree Conjugate of labeling is verified and must be 2 5 4 moles of Alexa Fluor 647 dye per mole of protein The conjugate is functionally qualified by immunocytochemistry and must show good nuclear staining with negligible background 56 Purchaser Notification Introduction Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 125 GST Use of the ProtoArray Human Protein or Yeast Proteome Microarray PPI Complete Kit for Epitope Tagged Proteins is covered under the licenses described below 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
15. low lysine Perform the biotinylation reaction at a 27 1 molar content or lysine residues ratio or higher You may express your protein as a are not readily available for fusion to a tag that contains lysine biotinylation Additional Protein impurities present Impurities may cause high background during biotinylated bands that undergo biotinylation probing Purify protein to remove impurities and observed perform biotinylation to ensure the absence of additional biotinylated bands Continued on next page 50 Troubleshooting Continued Control Array Results No signal Incorrect scanning or Be sure to scan the array at 635 nm or equivalent imaging and place the array in the slide holder such that the proteins on the array are facing the laser source If scanning is performed correctly the spots corresponding to the Alexa Fluor labeled antibody will be visible Use the recommended settings page 41 to obtain a good image Weak or no signal Presence of free biotin Purify the protein after biotinylation using the spin with biotinylated column supplied in the kit protein probe against the anti biotin antibody Weak or no signal Incorrect probing procedure Follow the recommended protocol for probing Be with biotinylated sure all incubations are performed at 4 C Prepare calmodulin kinase the PBST Blocking Buffer and Probing Buffer fresh probe as described on page 32 Do not allow the array to dry du
16. of free biotin from biotin labeled proteins The expected yield of the biotinylated protein is 50 60 While the biotinylation reaction is in progress prepare the spin columns for purification as follows 1 Remove 4 spin column collection tube assemblies from the module 2 Resuspend the Purification Resin by gently rocking the bottle until the resin is evenly suspended in the buffer Avoid mixing by shaking or vortexing 3 Transfer 800 ul of the Purification Resin into each of the 4 spin columns Centrifuge spin columns in a microcentrifuge at maximum speed 10 000 15 000 x g for 15 seconds at room temperature Note The fixed angle rotor of a microcentrifuge causes the resin in the spin column to have a low side and high side 5 Discard collection tubes and place spin columns in clean collection tubes supplied in the module Cap spin columns until sample application to prevent drying of resin which affects recovery 6 Proceed to the Purification Procedure next page Continued on next page 21 Purifying Biotinylated Protein Continued Purification Procedure 22 At the end of the biotinylation incubation period Step 8 page 20 purify the biotinylated samples using the spin columns prepared as described on the previous page 1 Load each of the 4 biotinylation reactions Step 8 page 20 onto 4 spin columns containing the purification resin prepared as described on previous page Load samples onto the m
17. open in the dark for 30 60 minutes at room temperature to dry the arrays Make sure the array is completely dry there should be no translucent areas Scan the array using a fluorescence microarray scanner see page 41 for details After scanning and saving an image of each array analyze results to identify positive interactors For more details see page 42 1 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 42 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the control array Analyze data using the guidelines on page 44 to determine significant signals with the Array Control Protein and your biotinylated protein probe Note An example of the expected results obtained after probing the Control Arrays is shown on page 46 For troubleshooting see page 50 After confirming the appropriate interactions on the Control Arrays proceed to Probing the ProtoArray Human or Yeast Microarrays next page At the end of probing experiments clean the Array Chambers properly and rinse with sterile water before re using the chambers 35 Probing the ProtoArray Human or Yeast Microarrays Introduction Materials Needed Important Experimental Outline Important Guidelines 36 After using the ProtoArray Control Protein Microarray nc to verify the q
18. printed on the array Note The yeast proteins printed on the array are derived from the Snyder collection Zhu et al 2001 For information on obtaining the yeast clone corresponding to the potential protein identified on the array contact Technical Support page 54 45 Expected Results Control Array Probing Results 46 Results obtained after probing the ProtoArray Control Protein Microarray nc v4 0 with the Array Control Protein i e BioEase V5 tagged biotinylated calmodulin kinase are shown below Image showing calmodulin Cmd1 signal when probed with the Array Control Protein Array Image Boxed Area shown in detail lee Aldxa Fluor Ab Je ajomatp pal Alexa Fluor Biotin Anti V5 Ab Biotin Control Ab Ab e Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities e Anti biotin Ab signal Biotinylated proteins bind to the Anti biotin antibody printed on the microarray Note The Array Control Protein contains an N terminal BioEase and V5 epitope tag The BioEase tag facilitates in vivo biotinylation of the protein during expression e Biotin Ab signal A biotinylated anti mouse antibody is printed on the microarray The Streptavidin Alexa Fluor 647 conjuga
19. the ProtoArray Blocking Buffer included in the kit without the addition of BSA Improper washing To obtain the best results perform the recommended washing steps Prepare the Probing Buffer fresh as described on page 32 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array as described on page 35 before before scanning scanning High probe concentration Decrease the probe concentration to 5 ug ml or decrease the incubation time Streptavidin Alexa Fluor Probe a human or yeast array using only the 647 Conjugate cross Streptavidin Alexa Fluor 647 Conjugate without reactivity the protein probe to detect cross reactivity with the conjugate only Uneven background See previous page for See previous page for details details Signal Due to The following yeast protein produces a significant signal with the Streptavidin Interaction with Alexa Fluor 647 conjugate For a list of proteins that may interact with Alexa Detection Reagent Fluor 647 conjugated streptavidin see the ProtoArray Central Portal ORF Name YGL062W Note that the signal does not indicate a positive interaction The array yeast protein is biotinylated based on the E motif database therefore it produces signal with Streptavidin Alexa Fluor 647 conjugate For more information on the E motif database visit http db yeastgenome org cgi bin SGD protein getDomain sgdid 50003030
20. the best detection results with reagents included in the ProtoArray Biotinylation Assessment Module follow these guidelines e Usea single clean container for each blot e Avoid touching the working surface of the membrane even with gloves e Avoid cross contamination of system solutions especially with the alkaline phosphatase substrate solution e Perform all washing blocking and incubation steps on a rotary shaker rotating at 1 revolution second e Add solutions to the trays slowly at the membrane edge to avoid bubbles forming under the membrane Decant from the same corner of the dish to ensure complete removal of previous solutions Prepare the solutions for analyzing 2 nitrocellulose membranes using the reagents included in the kit as described below For Nitrocellulose Membrane Blocking Solution Ultra filtered Water Western Blocking Solution A Western Blocking Solution B Total Volume Streptavidin AP Streptavidin AP Conjugate Solution 1 4000 Blocking Solution above Wash Solution Ultra filtered Water 150 ml Western Washing Buffer 16X 10 ml Total Volume 160 ml Chemiluminescent Chemiluminescent Substrate 4 75 ml Substrate Chemiluminescent Substrate Enhancer 0 25 ml Total Volume 5ml Continued on next page 27 Assessing Protein Biotinylation Continued Western Detection Protocol Assessing Biotinylation 28 1 10 Place each membrane in 10 ml of the Blocking So
21. treated at 3 1 molar ratio 25 fmoles Lane 8 Calmodulin kinase treated at 9 1 molar ratio 25 fmoles Lane 9 Calmodulin kinase treated at 27 1 molar ratio 25 fmoles Lane 10 BSA Control Protein treated at 9 1 molar ratio 25 fmoles Interpreting Results Compare the band intensities of your biotinylated protein to the BSA Control Protein and Biotinylation Gel Standard as described below to select a properly biotinylated protein probe 3 5 biotin molecules protein The BSA Control Protein 25 fmoles lane 10 above is modified with 3 5 biotin molecules per polypeptide Loading 25 fmoles BSA Control Protein is equivalent to loading 75 125 fmoles biotin The band intensity of 25 fmoles BSA Control Protein is approximately similar to the band intensity of 100 fmoles Biotinylation Gel Standard lane 3 above For a protein with average lysine content 8 biotinylating at a molar ratio of 9 1 usually incorporates 3 5 biotin molecules protein The band intensity of 25 fmoles of protein probe biotinylated at 9 1 lane 8 above should be approximately similar to the band intensity of BSA Control Protein lane 10 above or 100 fmoles Biotinylation Gel Standard lane 3 above Based on the biotinylation results of the example shown in the gel you can use calmodulin kinase biotinylated at 9 1 molar ratio for probing experiments 29 Probing the ProtoArray Control Protein Microarrays Introduction ProtoArray PP
22. 2 Continued on next page 14 Experimental Overview Continued Workflow The experimental workflow for probing the ProtoArray Human Protein or Yeast Proteome Microarray nc with your in vitro biotinylated probe is shown below Purify Protein Probe Quantitate Protein Do have enough protein Biotinylate with ProtoArray Mini Biotinylation Kit using 3 biotin ratios Purify Biotinylated Protein Quantitate Protein enough protein Purchase ProtoArray Mini No Biotinylation Kit YES Biotinylated Assess Biotinylation Gel Standard Biotinylation OK NO NO YES crouch Quantitate Probe Control Array 1 with Probe Control Array 2 with Scan 2 Protein best biotinylated protein Array Control Protein YES Contact Technical Service Do get an anti biotin signal Do you see control spots Spin column or dialyze NO See Trouble Shooting YES YES Probe Proteome Array 15 Methods Preparing the Protein Probe Introduction Protein Quality Amount of Protein 16 Before using the ProtoArray Human Protein or Yeast Proteome Microarray PPI Kit you need your purified protein of interest to probe the microarray You may purify proteins using any method of choice You can use proteins purified from E coli yeast cells or higher eukaryotes to probe the ProtoAr
23. 647 dye Built in controls are printed on each array to control for background and detection Arrays are compatible with most commercially available fluorescence microarray scanners Since most of the human and yeast proteins printed on the microarray contain a GST Glutathione S Transferase fusion tag and some proteins also contain polyhistidine 6x tag do not use an anti GST antibody or anti polyhistidine antibody for detecting interactions on a ProtoArray Human or Yeast Protein Microarray nc We strongly recommend that you probe the ProtoArray Human or Yeast Protein Microarray nc with only your detection reagent to detect signals resulting due to interactions between the detection reagent and proteins printed on the array The ProtoArray Microarrays are not compatible for use with Alexa Fluor 555 or Cy3 dyes Use of these fluorescent dyes results in high background on the array as the nitrocellulose surface has high intrinsic fluorescence at the wavelength used to visualize Alexa Fluor 555 or Cy3 dyes This manual provides the following information An overview of the ProtoArray Human Protein Yeast Proteome and Control Protein Microarrays Guidelines and instructions to in vitro biotinylate your protein probe Instructions to probe the ProtoArray Microarray with your protein probe Guidelines to perform data analysis Expected Results and Troubleshooting Description of Kit Components Components of th
24. Applications System Overview _ gt Block 1 hr add biotin labeled protein 90 min Introduction The ProtoArray Human Protein and Yeast Proteome Microarray PPI protein protein interaction Kits for Biotinylated Proteins allow rapid and efficient detection of human or yeast protein protein interactions using a biotinylated protein probe of interest The ProtoArray Human Protein Microarray nc contains thousands of purified human proteins while the ProtoArray Yeast Proteome Microarray nc contains gt 4000 purified yeast proteins from Saccharomyces cerevisiae In both cases the proteins are printed in duplicate on a nitrocellulose nc coated glass slide See below for an overview of the system ProtoArray Human Protein and Yeast Proteome Microarrays allow you to e Detect novel protein protein interactions e Validate previously observed protein protein interactions e Confirm positive interactions using the identified interacting protein on the array as a probe in reciprocal experiments e Test various experimental conditions for your protein protein interactions To use the ProtoArray Human Protein or Yeast Proteome Microarray PPI Kits you will e In vitro biotinylate your protein of interest using the reagents supplied in the kit e Use the biotinylated protein to probe the ProtoArray Control Protein Microarray nc to verify protein biotinylation and probing conditions e Probe the ProtoArray Hu
25. Array Human Protein Microarray nc to detect the interacting protein calmodulin kinase Then perform the reciprocal interaction with another human microarray using calmodulin kinase as the probe to detect the interacting protein calmodulin The ability to observe reciprocal interactions indicates that the proteins maintain a proper folded state on the array ProtoArray Yeast Proteome Microarray Introduction Yeast Proteome Microarray Specifications Array Specifications The ProtoArray Yeast Proteome Microarray nc v1 1 is a high density protein microarray containing the majority of proteins from S cerevisiae Each S cerevisiae open reading frame ORF is expressed as an N terminal GST 6xHis fusion protein purified and printed in duplicate on a nitrocellulose coated glass slide This section provides details about the yeast proteome microarray including array specifications and preparation of proteins Note The ProtoArray Yeast Proteome Microarray PPI Complete Kit includes 2 ProtoArray Yeast Proteome Microarrays The specifications for the ProtoArray Yeast Proteome Microarray nc v1 1 are listed below Dimensions 1 inch x 3 inch 25 mm x 75 mm Material Glass slide coated with nitrocellulose membrane Membrane Size 20 mm x 60 mm Membrane Properties Thickness 15 20 um Pore Size 0 2 um Each microarray has a barcode for tracking samples The barcode is also used to retrieve array specific information fr
26. DS Sample Buffer to a microcentrifuge tube Add 100 ul 10X NuPAGE Reducing Agent to the tube to generate 1X Gel Loading Buffer Transfer 40 ul Biotinylation Gel Standard to a microcentrifuge tube Add 4 ul 10X NuPAGE Reducing Agent and 36 ul 1X Gel Loading Buffer from Step 2 to obtain a 10 fmoles ul stock total volume 80 ul Starting with the 10 fmoles ul stock prepare 2 fold serial dilutions of the Biotinylation Gel Standard to obtain 5 fmoles ul 2 5 fmoles ul 1 25 fmoles ul and 0 625 fmoles ul stocks For each dilution dilute the standard with 1X Gel Loading Buffer to a final volume of 40 pl Heat the samples at 70 C for 10 minutes Load 20 ul sample on a NuPAGE Novex 4 12 Bis Tris Gel as described on page 26 Using these samples allows analysis of 200 fmoles 100 fmoles 50 fmoles 25 fmoles and 12 5 fmoles of the Biotinylated Gel Standard Continued on next page Assessing Protein Biotinylation Continued Note Formula for Generating Stock Solution Preparing Biotinylated Protein Samples for SDS PAGE A formula is included below for your convenience to generate a stock solution for each of your protein samples after column purification If you are an experienced user and are familiar with protein molar calculations you may use your own method for calculation Use the formula below to calculate the final volume of the sample to generate a 200 fmoles ll stock solution from 1 ul of co
27. I Buffer Modules ProtoArray Application Kit Materials Needed Important 30 The ProtoArray Control Protein Microarray nc allows you to verify in vitro biotinylation labeling and probing conditions Probe the ProtoArray Control Protein Microarrays prior to probing the ProtoArray Human Protein or Yeast Proteome Microarrays Instructions are provided in this section to probe the ProtoArray Control Protein Microarrays supplied with the kit The ProtoArray PPI Buffers Module A and B supplied with the complete kits include qualified reagents for blocking washing and detection during the microarray probing procedure The pre made buffers provide consistent results and eliminate the time required to prepare reagents ProtoArray PPI Buffer Module B also includes HybriSlip cover slips that hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents Array Chambers are also included in the module for washing the microarrays The ProtoArray Protein Protein Interaction Application Kit includes ProtoArray PPI Buffer Modules A and B and the Alexa Fluor detection reagent The use of application kit provides consistent results and eliminates the time required to prepare reagents The ProtoArray PPI Buffer Modules A and B include qualified reagents for blocking washing and probing during the microarray probing procedure see above Before using the ProtoArray
28. PPI Buffer Modules A and B and the Streptavidin Alexa Fluor 647 Conjugate only You will need to purchase a ProtoArray Human Protein Control Protein or Yeast Proteome Microarray nc separately from Invitrogen before performing a microarray screening experiment 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 will also use the portal to retrieve ProtoArray Lot Specific information see page 42 which is required for analysis of the array data and identification of statistically significant interactions The ProtoArray Prospector software quickly analyzes the microarray data acquired from the image 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 free of charge to ProtoArray users and are accessible online at the ProtoArray Central Web Portal To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click on Online Tools tab ProtoArray Human Protein Microarray Introduction Human Protein Microarray Specifications Array Specifications The ProtoArray Human Protein Micr
29. ain the best results and avoid any damage to the array or array proteins always handle the ProtoArray Microarrays with care using the following guidelines Always wear clean gloves while handling microarrays Do not touch the surface of the array to avoid any damage to the array surface resulting in uneven or high background Maintain the array and reagents at 2 8 C during the experiment To prevent condensation on the array that may reduce protein activity or alter spot morphology allow the mailer containing the array to equilibrate at 4 C for at least 15 minutes prior to removing the array from the mailer and immersing the array immediately in blocking solution equilibrated at 4 C Perform array experiments at a clean location to avoid dust or contamination and filter solutions if needed particles invisible to eyes can produce high background signals and cause irregular spot morphology Avoid drying the array during the experiment and ensure the array is completely covered with the appropriate reagent during all steps of the protocol Always dry the array prior to scanning and scan the array on the same day at the end of the experiment Do not dry the array using compressed air or commercial aerosol sprays Avoid exposing the array to light after probing with Streptavidin Alexa Fluor 647 conjugate Probes for Control Use the following biotinylated proteins to probe the ProtoArray Control Arrays Protein Microarrays Biotin
30. 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 diagnostic 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 c
31. bing procedure prior to probing the ProtoArray Human Protein or Yeast Proteome Microarray nc Two control arrays are included in each kit probe one array with your biotinylated protein probe to allow you to assess biotinylation quality and probe the second array with the Array Control Protein supplied in the kit biotinylated calmodulin kinase to validate assay conditions and demonstrate a known protein protein interaction between calmodulin kinase and yeast calmodulin Cmd1p Ybr109C For specifications and more details on the ProtoArray Control Protein Microarray see page 11 To detect protein protein interactions on the ProtoArray Human Protein or Yeast Proteome Microarray nc the protein probe must contain a label or tag to visualize the interaction of the probe with array proteins The extremely high affinity of the biotin streptavidin interaction makes biotin protein conjugation an attractive method for probe labeling The ProtoArray Mini Biotinylation Module provides a simple and efficient method to biotinylate small amounts of your protein probe using water soluble Biotin XX sulfosuccinimidy l ester The module includes sufficient reagents to biotinylate your protein probe at 3 molar ratios page 17 The biotinylated protein probe is detected using streptavidin conjugated to the fluorescent dye Alexa Fluor 647 see next page providing signal amplification and increased sensitivity After in vitro biotinylating the pr
32. ctions are provided in this section to prepare samples for SDS PAGE with NuPAGE Gels If you are using Tris Glycine gels prepare samples as directed on pages 24 25 Run gels using the buffers and conditions recommended by the manufacturer Continued on next page 23 Assessing Protein Biotinylation Continued Materials Needed Preparing Biotinylation Gel Standard For SDS PAGE 24 ProtoArray Biotinylation Assessment Module supplied with the complete kit Biotinylation Gel Standard supplied with the complete kit Aliquot of purified biotinylated protein probe and BSA step 6 page 22 1X NuPAGE LDS Sample Buffer supplied with the complete kit 10X NuPAGE Reducing Agent supplied with the complete kit 2 NuPAGE Novex Bis Tris Gels page x NuPAGE MES or MOPS SDS Running Buffer page x NuPAGE Antioxidant page x Nitrocellulose membranes page x Electrophoresis and blotting apparatus page x Deionized water Heating block set at 70 C Appropriate staining container for Western blotting Molecular weight markers page x Prepare the following dilutions of the Biotinylation Gel Standard to generate a standard curve for SDS PAGE Each ul of the Biotinylation Gel Standard contains 20 fmoles of biotin conjugated to BSA and is used for assessing biotinylation 1 Thaw the Biotinylation Gel Standard 1X NuPAGE LDS Sample Buffer and 10X NuPAGE Reducing Agent Transfer 900 ul 1X NuPAGE L
33. d Analysis soe prn e A DAR EA RA ERA E E RE E cok 42 Expected Resulta il iia ae S a N aE EE q Dota GA RA ERE tne 46 Troubleshooting evi idad aliadas 50 APRENDA 54 Technical Supports asias 54 Product QUAN ita 55 Purchaser Notifica ti orn 20 a e aiii 57 Referentes initial it 59 iii iv Kit Contents and Storage Types of Kits This manual is supplied with the following kits Product Catalog no ProtoArray Human Protein Microarray PPI Complete Kit v4 0 PAH0524011 for biotinylated proteins ProtoArray Yeast Proteome Microarray PPI Complete Kit v1 1 PA0121011 for biotinylated proteins ProtoArray Protein Protein Interaction Application Kit PA011 for biotinylated proteins Kit Components The ProtoArray PPI Kits for Biotinylated Proteins include the following components For a detailed description of the contents of each component see pages vi ix Note Catalog nos PAH0524011 and PA0121011 include two ProtoArray Human Protein or Yeast Proteome Microarrays as appropriate and two ProtoArray Control Protein Microarrays Component Catalog no PAH0524011 PA0121011 PA011 ProtoArray Human Protein Microarray nc v4 0 y ProtoArray Yeast Proteome Microarray nc v1 1 ProtoArray Control Protein Microarray nc v4 0 Array Control Protein Streptavidin Alexa Fluor 647 Conjugate ProtoArray PPI Buffer Module A ProtoArray PPI Buffer Module B ProtoArray Mini Biotinylation Module ProtoArray
34. e Microarray nc use the identified interacting protein from the array as a probe for to probe another ProtoArray Yeast Proteome Microarray to confirm the reciprocal interaction For an example of reciprocal interaction see page 49 ProtoArray Yeast Proteome Microarrays are produced using the same rigorous production and pre printing and post printing quality control procedures used to produce the human protein microarrays see page 8 For detailed product qualification see page 55 Continued on next page ProtoArray Control Protein Microarray Introduction Control Microarray Specifications Control Array Specifications The ProtoArray Control Protein Microarray nc contains protein interactors and various controls printed on a nitrocellulose coated glass slide The Control Protein Microarrays allow you to validate probing procedures prior to probing the ProtoArray Human Protein or Yeast Proteome Microarray Details about the ProtoArray Control Protein Microarray are described in this section The specifications for the ProtoArray Control Protein Microarray are listed below Dimensions 1 inch x 3 inch 25 mm x 75 mm Material Glass slide coated with nitrocellulose membrane Membrane Size 20 mm x 60 mm Membrane Properties Thickness 15 20 um Pore Size 0 2 um Each microarray has a barcode for tracking samples The barcode is also used to retrieve array specific information from the ProtoArray
35. e ProtoArray PPI Kits ProtoArray Human Protein and Yeast Proteome Microarrays The ProtoArray Human Protein or Yeast Proteome Microarray PPI Complete Kits for Biotinylated Proteins include the following major components e The ProtoArray Human Protein or Yeast Proteome Microarray a high density protein microarray that allows you to screen your protein of interest protein probe against thousands of human proteins or the Saccharomyces cerevisiae proteome respectively e The ProtoArray Control Protein Microarray nc and the Array Control Protein for verification of the probing conditions and background levels e The ProtoArray Mini Biotinylation Module for in vitro biotinylation of the protein probe e The ProtoArray Biotinylation Purification Module for removing free biotin from your biotinylation reaction e The ProtoArray Biotinylation Assessment Module for validation of the level of biotinylation achieved in the protein probe e The ProtoArray PPI Buffer Modules A and B contain pre made qualified reagents for performing the blocking and washing steps during probing e The Streptavidin Alexa Fluor 647 Conjugate for detection The ProtoArray Human Protein and Yeast Proteome Microarrays are high density protein microarrays containing human or S cerevisiae proteins respectively The ProtoArray technology is based on the yeast protein microarray technology developed by Zhu et al 2001 to detect molecula
36. e ProtoArray Human Protein or Yeast Proteome Microarrays with a fluorescence microarray scanner at 635 nm 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 enclosure on the scanner Place the ProtoArray Human Protein or Yeast Proteome Microarray nc in the holder such that the nitrocellulose coated side of the array faces the laser source and the barcode on the array is closest to the outside of the instrument Close the microarray enclosure on the scanner Set the following settings to image the microarray 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 Note The image should have very few saturated spots white Adjust settings such that the Alexa Fluor Ab spots are at or near the pixel saturation 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
37. e from Step 9 to the chamber Incubate the chamber for 30 minutes on ice in the dark cover the ice bucket Decant the buffer Invert the chamber on paper towels for a few seconds to drain excess buffer Slowly add 25 ml Probing Buffer onto the chamber wall while keeping the chamber on ice Avoid pipetting buffer directly onto the array surfaces Incubate the array in Probing Buffer for 1 minute on ice Decant the buffer Drain excess buffer by inverting chamber on paper towels for a few seconds Repeat Steps 12 13 two more times using 25 ml Probing Buffer each time Proceed to Drying the Arrays next page Continued on next page Probing the ProtoArray Control Protein Microarrays Continued Drying the Arrays Data Analysis Cleaning the Chamber 1 Remove the arrays from the chamber at the end of the probing procedure Tap one edge of each array gently on a laboratory wipe for a few seconds to drain any buffer Place the arrays in a slide holder or a sterile 50 ml conical tube if you do not have a slide holder in a vertical orientation Ensure that the arrays are properly placed and secure in the holder to prevent any damage to the arrays during centrifugation Centrifuge the arrays 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 arrays in a slide box and keep the box with the lid
38. ed as described on the previous page e Review the experimental outline on page 18 Dissolve biotin XX sulfosuccinimidyl ester in cold water immediately before use and add the biotin reagent to the protein within 5 minutes of initial resuspension of the reagent in water The ProtoArray Mini Biotinylation Kit contains 100 ug 150 nmoles of lyophilized biotin XX sulfosuccinimidyl ester Prepare and dilute the biotin reagent for biotinylating proteins at 3 molar ratios see Fig B page 18 as follows 1 To three 0 5 ml microcentrifuge tubes add the following on ice Tube 1 2 3 Chilled Sterile Water 8 ul 8 ul 2 To 100 ug 150 nmoles of lyophilized biotin XX sulfosuccinimidy ester add 10 ul cold sterile water 3 Mix well by pipetting up and down Be sure to completely dissolve the contents of the vial Centrifuge briefly in a microcentrifuge Transfer entire contents 10 jul of the tube into Tube 1 from Step 1 4 Transfer 4 ul from Tube 1 to Tube 2 containing 8 pl sterile water Mix well and centrifuge briefly in a microcentrifuge 5 Transfer 4 ul from Tube 2 to Tube 3 containing 8 ul sterile water Mix well and centrifuge briefly in a microcentrifuge The final concentration and volume in each tube is listed below Tube 1 2 3 Final Biotin Reagent 15 nmole ul 5 nmole ul 1 67 nmole ul Concentration Final Volumes 6 ul 8 ul 12 ul Use for protein molar ratio 27 1 9 1 3 1 6 Add x pl see f
39. er the array barcode in the Input Barcode Number s box Click on the Search button Continued on next page Data Acquisition and Analysis Continued ProtoArray Central continued 4 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 human proteins on the array 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 designed for different applications For example ProtoArray Prospector uses the file HA10756 PPLPAI to analyze data from PPI experiments performed on array from lot HA10756 Slide Information File LotNumber_slide txt This file contains a listing of all barcodes associated with a specific lo
40. expressed as an N terminal GST 6xHis fusion protein in the yeast expression vector pEG KG Mitchell et al 1993 The identity of each clone is verified using 5 end sequencing and the expression of GST tagged fusion protein by each clone is confirmed with Western immunodetection using an anti GST antibody The proteins are then expressed and purified using high throughput procedures Briefly yeast stocks are initiated in growth media protein expression is induced with galactose and cell lysates prepared The proteins are purified using glutathione affinity chromatography eluted and purified proteins are used to spot the proteome microarray The purified yeast proteins are printed on nitrocellulose coated slides in a dust free temperature and humidity controlled environment to maintain consistent quality of microarrays The arrays are printed using an automated process on an arrayer that is extensively calibrated and tested for printing ProtoArray Microarrays Various proteins and controls are printed on each ProtoArray Yeast Proteome Microarray to allow you to verify background and detection conditions during probing For details see page 12 ProtoArray Yeast Proteome Microarrays are ideal for detecting reciprocal interactions since the microarrays are manufactured under highly controlled conditions to ensure maximum protein function Once you have identified a positive interaction using the ProtoArray Yeast Proteom
41. gloved hands or forceps Take care while inserting the array into the Array Chamber to avoid scratching the array surface TM Apply the probe solution and HybriSlip to the array as described in the manual To avoid drying of the membrane make sure the HybriSlip covers the membrane area of the array and adjust the HybriSlip if needed Probe or detection reagents Centrifuge the probe or detection reagents to contain precipitates remove precipitates prior to probing the array Human Protein or Yeast Proteome Array Results Poor biotinylation of protein See page 37 for details probe Low probe concentration Incorrect scanning or imaging Interaction conditions too stringent Perform probing with higher probe concentration or increase the incubation time Use the probe biotinylated at a higher molar ratio or perform biotinylation at a higher molar ratio Scan the array at 635 nm or equivalent and place the array in the slide holder such that the proteins on the array are facing the laser source Use the recommended settings page 41 to obtain a good image Decrease the number of washes Perform probing and washing in the absence of or in lower concentration of detergent or salts Continued on next page Troubleshooting Continued Human Protein or Yeast Proteome Array Results continued High background Improper blocking Prepare the PBST Blocking Buffer fresh as described on page 32 Do not use
42. grity function and activity Approximately 5000 human proteins printed on the ProtoArray Human Note Protein Microarray nc v4 0 were also present on the previous version of the product ProtoArray Human Protein Microarray nc v3 0 However not all proteins printed on ProtoArray Human Protein Microarray nc v3 0 are also printed on ProtoArray Human Protein Microarray nc v4 0 To obtain a list of ProtoArray Microarray nc v3 0 human proteins that are not printed on ProtoArray Microarray nc v4 0 contact Technical Support page 54 Continued on next page ProtoArray Human Protein Microarray Continued Controls Various proteins and controls are printed on each ProtoArray Human Protein Microarray to allow you to verify background and detection conditions during probing For details see page 12 Printing the The purified human proteins are printed on nitrocellulose coated slides in a Human dust free temperature and humidity controlled environment to maintain ProtoArray consistent quality of the microarrays The arrays are printed using an automated process on an arrayer that is extensively calibrated and tested for printing ProtoArray Microarrays Maintaining ProtoArray Human Protein Microarrays are produced using rigorous Stringent Quality production and quality control procedures with an integrated data Control management system to ensure consistent results with every array and maximize inter and intra l
43. iddle of the resin 2 Place the spin column in the microcentrifuge with the high side of the resin pointing up 3 Centrifuge the spin columns in a microcentrifuge at maximum speed 10 000 15 000 x g for 1 minute at room temperature 4 The purified biotinylated protein is in the collection tube The eluate volume is 50 100 ul and recovery is 50 60 5 Save a small aliquot for protein estimation and assessing biotinylation next page Store remaining samples at 4 C 20 C or 80 C depending on your protein Use the samples to probe the ProtoArray Human Protein or Yeast Proteome Microarray nc after assessing the level of biotinylation 6 Determine the final protein concentration of the biotinylated protein samples with 5 10 ul of the column eluate using a method of choice 7 Proceed to Assessing Protein Biotinylation next page Assessing Protein Biotinylation Introduction ProtoArray Biotinylation Assessment Module Experimental Outline Note Pre Cast SDS PAGE Gels Due to the differences in protein e g lysine residues the level of biotinylation on each protein varies To obtain the best results with the biotinylated protein probe for use with the ProtoArray Human Protein or Yeast Proteome Microarray nc it is important to determine and assess the level of biotinylation for your protein sample Instructions for assessing protein biotinylation with Western blotting and chemiluminescent detection us
44. ile from www invitrogen com protoarray as described on page 42 Expression and Almost all clones used to generate the human protein collection are entry clones Purification of consisting of a human ORF cloned into a Gateway entry vector Each entry clone Human Proteins is subjected to a LR recombination reaction with a Gateway destination vector to generate an expression clone The expression clone is then used to express the protein as an N terminus GST fusion protein in some clones using the Bac to Bac Baculovirus Expression System available from Invitrogen For more information on the Bac to Bac Baculovirus Expression System visit www invitrogen com The LR reaction mix obtained after performing the LR reaction is transformed into competent DH10Bac E coli to generate a recombinant bacmid The high molecular weight recombinant bacmid DNA is isolated and transfected into Sf9 insect cells to generate a recombinant baculovirus that is used for preliminary expression experiments After the baculoviral stock is amplified the high titer stock is used to infect Sf9 insect cells for expression of the recombinant protein of interest After verifying that each clone expresses a protein of the expected molecular weight by western blotting the proteins are expressed and purified using high throughput procedures The expressed proteins are purified by affinity chromatography under conditions optimized to obtain maximal protein inte
45. image Note Examples of expected image scans of control human and yeast arrays are shown on pages 46 49 Open the microarray enclosure and remove the microarray from the holder Proceed to download lot specific information available on the ProtoArray Central Portal next page To orient the results obtained from the GAL file and ProtoArray Prospector with the array image position the microarray image such that the barcode is at the bottom of the image In this orientation the top left corner of the microarray image is Block 1 41 Data Acquisition and Analysis Introduction Important GAL File ProtoArray Central 42 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 Use the lot specific information to acquire and analyze the data to identify protein protein interactions Note To familiarize yourself with the array and subarray layout you may also download a file showing the subarray layout from ProtoArray Central To access the file go to www invitrogen com protoarray and click on Online Tools 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 d
46. in Molecules Protein 3 1 1 2 9 1 3 5 27 1 10 15 A 9 1 molar ratio results in a biotinylation efficiency of 3 5 biotin molecules per polypeptide for average proteins Proteins with few accessible lysine residues may label poorly with 9 1 molar ratio and may require a 27 1 molar ratio for better biotinylation Proteins with more lysine residues may over biotinylate with 9 1 molar ratio and produce better probe quality with a 3 1 molar ratio Continued on next page 17 In Vitro Biotinylation Continued Experimental The figure below outlines the steps required for in vitro biotinylation of your Outline protein probe using the ProtoArray Mini Biotinylation Module 1 MNaHCOz A 2 ul 2ul 2 yl 2ul 1 Aliquot 20 ul of 2 5 ug ul purified protein into 3 tubes and 20 ul of 2 5 ug l BSA control into 1 tube Add 2 ul of 1 M NaHCO into each tube gt lt gt 50 ug protein 50 ug protein 50 ug protein 50 ug BSA control in 20 wl vol in 20 wl vol in 20 ul vol in 20 ul vol B A N ATA SS 2 Calculate the amount of biotin 100 pg 150 anaes reagent x ul to add to your Biotin XX 10 ul H3 protein using the formula provided on the next page 8 ul 8 ul wa hA id bA 3 Prepare 3 fold serial dilutions 15 nmoles ul 5 nmoles pl 1 67 nmoles ul of biotin reagent Biotin reagent Biotin reagent Biotin reagent C xul x ul 1 35 pul xpl 4 Add x ul of biotin reagent from each serial dilution to the appropriate p
47. inase phosphorylates the Control Kinase Substrate CAMK2A Calcium calmodulin dependent protein kinase II alpha A human protein kinase that is used as a positive control for the small molecule profiling application 12 ProtoArray Control Protein Microarray Continued The yeast calmodulin protein Cmd1p expressed as described on page 10 is printed on each microarray When probing the ProtoArray Control Protein ney Microarray nc with the Array Control Protein i e biotinylated yeast calmodulin kinase these proteins interact This interaction can be used to verify the reagents and procedures used to probe the human and yeast microarrays Maintaining The ProtoArray Control Protein Microarrays are produced using the same Stringent Quality rigorous production and pre printing and post printing quality control Control procedures used to produce the ProtoArray Human Protein and Yeast Proteome Microarrays page 8 In addition the control arrays are functionally qualified by probing the arrays with the Array Control Protein biotinylated yeast calmodulin kinase to detect the appropriate interaction with calmodulin For detailed product qualification see page 55 13 Experimental Overview Experimental The recommended experimental timeline is outlined below A detailed Timeline experimental workflow is shown on the next page Analyze Results Analyze Results Day
48. incomplete transfer Monitor the transfer of pre stained protein Western detection standard bands to determine the transfer efficiency Insufficient exposure time Increase the exposure time Incorrect gel used Use a suitable percentage gel to separate your protein of interest Only Biotinylated Poor or no biotinylation of Make sure that the biotinylation reaction was Gel Standard bands BSA Control protein and performed as described on page 20 using the visualized protein probe specified molar ratios and at pH 8 0 Check that the calculations and serial dilutions were performed correctly Check the pH after addition of sodium bicarbonate buffer to ensure the pH of the sample is pH 8 0 Add the Biotin XX reagent to the protein probe within 5 minutes as the reagent degrades quickly in an aqueous solution Only Biotinylated Poor or no biotinylation of Make sure the protein is in a buffer that does not Gel Standard and your protein probe contain any primary amines such as ammonium Biotinylation ions Tris glutathione imidazole or glycine Control Protein The Biotin XX ickly i bands visualized e Biotin XX reagent degrades quickly in an aqueous solution and must be added to the protein probe within 5 minutes Make sure that the biotinylation reaction was performed as described on page 20 using the specified molar ratios and at pH 8 0 Check that the calculations and serial dilutions were performed correctly Protein has
49. ing the ProtoArray Biotinylation Assessment Module are provided in this section The ProtoArray Biotinylation Assessment Module provides an efficient and sensitive method of assessing the level of biotinylation and includes a Biotinylation Gel Standard Assessment is performed by SDS PAGE of biotinylated protein samples and the Biotinylated Gel Standard Western transfer to nitrocellulose membranes see Note below detection with Streptavidin AP conjugate and visualization using a chemiluminescent substrate The band intensities of the biotinylated protein samples are compared to the Biotinylation Gel Standard to assess the level of biotinylation 1 Perform SDS PAGE using biotinylated protein samples and the Biotinylation Gel Standard from the kit Transfer proteins to nitrocellulose membrane Perform Western chemiluminescent detection with Streptavidin AP conjugate Verify and assess the level of biotinylation for your protein probe We recommend using nitrocellulose membranes for Western detection to assess protein biotinylation Our results with ProtoArray Biotinylation Assessment Module have demonstrated lower sensitivity and higher background using PVDF membranes for Western detection A large variety of pre cast gels for SDS PAGE are available from Invitrogen For details visit our web site at www invitrogen com or contact Technical Support page 54 We recommend using NuPAGE Novex Bis Tris Gels and instru
50. invitrogen ProtoArray Protein Microarray PPI Kits for Biotinylated Proteins For detecting protein protein interactions PPI using a human or yeast protein microarray and a biotinylated protein Catalog nos PA011 PA0121011 and PAH0524011 Version D 18 October 2006 25 0786 ii Table of Contents Fable Of Contents miii ida Se had sada ese aa a sag boca CASA a ade a aa a Ei ses A IAE Aseos iii Kit Contents add Storage iio ata v Accessory POUR S cise A R E R sistaudebebseee ouences x INtrOdUCHON e 1 QV CL VICW viere eiiie AAA Bde el etek i deters tele AAA A 1 Description Of Kit Components iii A Aid a bd E AA E ansty 3 ProtoArray Human Protein Microarray A A s Resonant 6 ProtoArray Yeast Proteome Microarray is id da ai 9 ProtoArray Control Protein Microarray A A AA AA A A 11 Experimental OVEIVIE Wal Ad EER A E EA 14 Memos y E 16 Preparing the Protein Probe lt ccasscceeciescneseratestsvcsoncssvehsdenobeanensnatstdentasnonsechdedensssseneuerddesstecbenpastshsedeesueesnsnestbese 16 In Vitro Biotinylationiwiiites tan dias ra innana ee ain ddidiaaaiadabiatus dim audi a RER ESA diets 17 Purifying Biotinylated Protesis ieta etena ae E a e E E eE a E EEN EO EATE SEE oS 21 Assessing Protein Biotin ylation seie an a e E E AE a estates 23 Probing the ProtoArray Control Protein Ni aia 30 Probing the ProtoArray Human or Yeast MicroarTayS ooo errar 36 SCANMIN EG ATTAYS vse AE dated Baa ete A A EE AA AN E A 39 Data Acquisition an
51. ion to biotinylate your protein probe with the ProtoArray Mini Biotinylation Module supplied with the ProtoArray complete kits See the next page for an outline of experimental steps To obtain the best results with the ProtoArray Human or Yeast PPI Kits use the ProtoArray Mini Biotinylation Module supplied with the kit to biotinylate your protein probe The module is specifically formulated to biotinylate small amounts of your protein probe at 3 different molar concentrations of biotin to protein and includes a control protein to verify biotinylation efficiency The biotin XX sulfosuccinimidyl ester sodium salt included in the kit is water soluble and readily reacts with the amine group of lysine residues to yield a biotin moiety covalently attached to the protein probe via two aminohexanoic chains XX This 14 atom spacer has been shown to enhance the ability of the biotin moiety to bind to avidin Molecular Formula C2H 1N5NaO 0S gt Molecular Weight 669 74 Da Structure of biotin XX sulfosuccinimidyl ester sodium salt No A Pe NH D H Ho f o c O oO so Na LS Y Y 3 NH CH5 5 Cc NH CH 5 CON O We recommend biotinylating your protein probe at 3 molar ratios of 3 1 9 1 and 27 1 biotin protein probe in the final biotinylation reaction mixture Biotinylating the protein probes at these molar ratios typically incorporates the following number of biotin molecules per protein Molar Ratio Biot
52. is a registered trademark of Molecular Devices Corporation ScanArray is a registered trademark of PerkinElmer Inc AlphaArray is a registered trademark of Alpha Innotech Corporation arrayWoRx is a registered trademark of Applied Precision LLC SpotLight is a trademark of TeleChem International Inc GeneChip is a registered trademark of Affymetrix Inc Microsoft is a registered trademark of Microsoft Corp Coomassie is a registered trademark of Imperial Chemical Industries PLC 59
53. lated You can also perform a densitometry scan See next page for an example of a Western blot Compare the band intensities of 3 different molar ratios of biotinylated protein samples from Step 9 above to the BSA Control Protein and Biotinylation Gel Standard on the blot Use the biotinylated protein sample that gives the best signal at the lowest biotinylation molar ratio to probe the control array page 30 and human or yeast array page 36 The next page shows results of a biotinylation experiment and provides guidelines on interpreting your biotinylation results To troubleshoot biotinylation problems see page 50 Continued on next page Assessing Protein Biotinylation Continued Expected Results An example of the Western blot of Biotinylation Gel Standard and a biotinylated protein probe calmodulin kinase is shown below Calmodulin kinase and Control Protein BSA were biotinylated as described in this manual 20 ul of sample was electrophoresed on a NuPAGE Novex 4 12 Bis Tris Gel using NuPAGE MES SDS Running Buffer The proteins were transferred to a 0 45 um nitrocellulose membrane and proteins were detected using chemiluminescent detection as described on page 28 using a 5 second exposure 123456 78910 Samples on the gel are Lane 1 SeeBlue Plus2 Pre Stained Standard 10 ul a Lanes 2 6 Biotinylated Gel Standard 200 100 50 25 and SS al 12 5 fmoles respectively Lane 7 Calmodulin kinase
54. lumn purified material for each of the 3 protein biotinylation reactions treated at 27 1 9 1 and 3 1 molar ratio and control BSA biotinylation reaction treated at 9 1 molar ratio You will need to know the protein concentration in mg ml and the approximate molecular weight for each protein sample The molecular weight of BSA used for control biotinylation reaction is 66 700 Da 5 x 10 x protein concentration mg ml final volume in ul MW Da MW is the molecular weight of the protein in Daltons Example If the protein concentration of your sample after column purification page 22 is 0 5 mg ml and the MW of your protein is 50 000 Da calculate the final volume as follows 5 x 10 x 0 5 50 pl 50000 In this example dilute 1 ul of each sample with 49 ul 1X Gel Loading Buffer to generate a 200 fmoles protein ul stock solution for each sample Prepare the following dilutions of the biotinylated protein sample and BSA Control Protein after column purification for assessing biotinylation 1 Prepare a 200 fmoles ul stock solution for each sample using the formula described above 2 From the 200 fmoles ul stock solution for each sample prepare the following dilutions e Dilute 1 ul of 200 fmoles ul solution from each sample with 39 ul 1X Gel Loading Buffer to generate a 5 fmoles ul sample total volume is 40 ul e Dilute 10 ul of 5 fmoles ul solution from each sample with 30 ul 1X Gel Loading Buffer to ge
55. lution in a staining container Incubate for 30 minutes on a rotary shaker set at 1 revolution sec Decant the Blocking Solution Rinse the membrane with 20 ml of water for 5 minutes then decant Repeat once Incubate the membrane in 10 ml of Streptavidin AP Solution 1 4000 for 30 minutes then decant Wash the membrane for 5 minutes with 20 ml of Wash Solution then decant Repeat 3 times Rinse the membrane with 20 ml of water for 2 minutes then decant Repeat twice Place the membrane on a sheet of transparency plastic Do not allow the membrane to dry out With a clean pipette evenly apply 2 5 ml of the Chemiluminescent Substrate to the membrane surface without touching the membrane surface Let the reaction develop for 5 minutes Blot the excess Chemiluminescent Substrate solution from the membrane surface with the filter paper Do not allow the membrane to dry out Cover the membrane with another clean piece of transparency plastic to prepare a membrane sandwich for luminography Expose an X ray film we recommend Kodak X OMAT AR films to the membrane sandwich for 5 60 seconds see next page for an example of the blot Note The alkaline phosphatase activated CDP Star produces a maximum light emission wavelength at 466 nm to 461 nm depending on the membrane environment of the reaction Proceed to assess the Western detection results below Verify that the protein sample and Control Protein BSA is biotiny
56. man Protein or Yeast Proteome Microarray nc with the biotinylated protein probe to detect protein protein interactions The ProtoArray detection protocol includes instructions to block the array probe the array with your biotinylated protein probe wash to minimize non specific interactions detect interactions using the Streptavidin Alexa Fluor 647 Conjugate dry scan the array to view results acquire the array image and analyze results see figure below For a detailed experimental workflow see page 15 IM so TITS vveove eevee gt Wash 10 min add Alexa Fluor 647 conjugated streptavidin Q e for detection 30 min Wash dry and scan 1 hr TTT TTS Yu vveeove Yu 0O Continued on next page Overview Continued Advantages Important Purpose of the Manual Using the ProtoArray Human Protein or Yeast Proteome Microarray PPI Kits to detect protein protein interactions offers the following advantages Provides a simple rapid and efficient method to identify protein interactions within a day Includes qualified reagents for in vitro biotinylation buffers and detection reagents for probing eliminating the need to prepare reagents Includes controls to verify biotinylation and Western detection protocols Allows screening of your protein of interest against thousands of human or yeast proteins Provides sensitive stable fluorescence detection using the Alexa Fluor
57. mount of reagents used accordingly PBST Blocking Buffer 1X PBS 1 BSA 0 1 Tween 20 1 Use reagents provided in the kit to prepare 30 ml PBST Blocking Buffer as follows ProtoArray Blocking Buffer 10X 3 ml 30 BSA 1 ml Deionized water to 30 ml 2 Mix well do not vortex and store on ice until use Probing Buffer 1X PBS 5 mM MgCl 0 5 mM DTT 0 05 Triton X 100 5 Glycerol 1 BSA 1 Use reagents provided in the kit to prepare 180 ml Probing Buffer as follows ProtoArray Probe Buffer 5X 36 ml 1M DTT 90 ul 1M MgCl 0 9 ml 30 BSA 6ml Deionized water to 180 ml 2 Mix well do not vortex and store on ice until use After preparing PBST Blocking Buffer and Probing Buffer immediately return the remaining 5X ProtoArray Probe Buffer 10X ProtoArray Blocking Buffer and 1 M MgCl to 4 C and 30 BSA and 1 M DTT to 20 C Continued on next page Probing the ProtoArray Control Protein Microarrays Continued Preparing the Array Control Protein biotinylated calmodulin kinase Probes Mix 12 ul of the Array Control Protein included in the complete kits with 120 ul of Probing Buffer Mix well do not vortex and store on ice until use Biotinylated Protein Probe You need 120 ul of the protein probe Use the biotinylated protein sample that gives the best signal on Western blot at the lowest biotinylation molar ratio and dilute the probe to 50 ug ml in Probing Buffer Mix well do not vortex and
58. n Fluorescent Proteins Anal Biochem 306 50 54 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 Mitchell D Marshall T and Deschenes R 1993 Vectors for the Inducible Overexpression of Glutathione S Transferase Fusion Proteins in Yeast Yeast 9 715 722 Predki P 2003 Functional Protein Microarrays Ripe for Discovery Curr Opin Chem Biol 8 8 13 Schweitzer B Predki P and Snyder M 2003 Microarrays to Characterize Protein Interactions on a Whole Proteome Scale Proteomics 3 2190 2199 Zhu H Bilgin M Bangham R Hall D Casamayor A Bertone P Lan N Jansen R Bidlingmaier S Houfek T Mitchell T Miller P Dean R A Gerstein M and Snyder M 2001 Global Analysis of Protein Activities Using Proteome Chips Science 293 2101 2105 2004 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 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 TM Cy5 is a trademark of Amersham Biosciences GenePix
59. nals or high background see Troubleshooting page 50 Scanning Arrays Introduction Materials Needed Experimental Outline Scanner Specifications Once you have probed the ProtoArray with your biotinylated protein scan the microarray using a fluorescence microarray scanner You need a fluorescence microarray scanner to scan the ProtoArray Human Protein or Yeast Proteome Microarray nc To acquire ProtoArray data from the image you will 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 The scanner specifications are listed below and recommended scanners are listed on the next page Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microarray and adjust settings if needed 1 2 3 4 Scan the microarray 5 Align grid over spots and use image analysis software to align features 6 Export and analyze results The fluorescence microarray scanner specifications required to image the ProtoArray Human Protein or Yeast Proteome Microarray nc are listed in the table below Array Compatibility Size Standard 1 x 3 or 25 mm x 75 mm microscope slides Thickness 1mm Detection Light and Detector Facing array Orientation Scanned Area 22 mm x 73 mm Focus Auto focus or adjustable 200 um
60. nerate a 1 25 fmoles ul sample total volume is 40 ul Heat the samples at 70 C for 10 minutes 4 Load 20 ul sample on a NuPAGE Novex 4 12 Bis Tris Gel as described on the next page The final amount for each sample is listed on the next page Continued on next page 25 Assessing Protein Biotinylation Continued Gel Electrophoresis 26 After preparing samples perform SDS PAGE You need 2 NuPAGE Novex Bis Tris mini gels for analysis Note You may load samples on one gel as shown in the example on page 29 if desired The recommended loading pattern and final amount for each sample is listed below Load 20 ul of each sample on the gel and 10 ul of a molecular weight protein standard For samples containing 25 fmoles Gel 1 Lanes 6 9 or 100 fmoles Gel 2 Lanes 6 9 of biotinylated protein or BSA use the 1 25 fmoles ul or 5 fmoles ul stock solutions Step 2 page 25 respectively Gel 1 Lane Sample Final Amount in Gel 1 Biotinylated Standard 200 fmoles biotin 2 Biotinylated Standard 100 fmoles biotin 3 Biotinylated Standard 50 fmoles biotin 4 Biotinylated Standard 25 fmoles biotin 5 Biotinylated Standard 12 5 fmoles biotin 6 Biotinylated protein 3 1 25 fmoles protein 7 Biotinylated protein 9 1 25 fmoles protein 8 Biotinylated protein 27 1 25 fmoles protein 9 Biotinylated BSA 9 1 25 fmoles protein 10
61. nteractions observed on the top left corner for the array is due to calmodulin printed on the microarrays as control spots The interaction of the yeast proteome calmodulin with the calmodulin kinase probe is shown in detail Continued on next page 48 Expected Results Continued Example of Reciprocal Interaction Example Showing High Background Demonstration of reciprocal interactions provides more confidence that the interactions observed most likely result from a direct protein protein interaction between the labeled protein probe and the array protein An example of a reciprocal interaction observed after probing the ProtoArray Yeast Proteome Microarray nc v1 0 is shown below Reciprocal interactions have been also been demonstrated with the ProtoArray Human Protein Microarray results not shown Biotinylated Calmodulin probe interacting with calmodulin kinase Cmk1p Biotinylated Calmodulin kinase probe interacting with calmodulin Cmd1p es Alexa_ Fluor Ab Biotin Ab gradient In this example the ProtoArray Control Protein Microarray nc was dried during the probing procedure producing high background 49 Troubleshooting Introduction The table below provides some solutions to possible problems you may encounter when using the ProtoArray Human Protein or Yeast Proteome Microarray PPI Complete Kit In vitro Biotinylation No signal after Poor or
62. nued ProtoArray Biotinylation Assessment Module The ProtoArray Biotinylation Assessment Module includes the following reagents Store at 4 C Note Sufficient reagents are included to perform 2 Western detections Western Blocking Buffer A Western Blocking Buffer B Western Washing Buffer 16X Chemiluminescent Substrate Chemiluminescent Substrate Enhancer Streptavidin Alkaline Phosphatase AP Conjugate Concentrated buffered saline solution containing detergent Concentrated Hammersten casein solution Concentrated buffered saline solution containing detergent Ready to use solution of CDP Star chemiluminescent substrate for alkaline phosphatase Nitro Block IT enhancer Supplied in 3 M NaCl 1 mM MgCh 0 1 mM ZnCl and 30 mM triethanolamine pH 7 6 The conjugate has 1600 2600 units per ml of alkaline phosphatase activity and a concentration from 0 75 1 2 mg ml Accessory Products Additional The table below lists additional products available separately from Invitrogen Products For more information about these products visit www invitrogen com or call Technical Support page 54 Product Quantity Catalog no ProtoArray Human Protein Microarray nc v4 0 1 array PAH052401 ProtoArray Yeast Proteome Microarray nc v1 1 1 array PA012101 ProtoArray Control Protein Microarray nc v4 0 1 array PA1007 ProtoArray Protein Protein Interaction Buffer Modules 1 kit PAO14 P
63. oArray Prospector software and manual are available free of charge to ProtoArray users To download the ProtoArray Prospector software or manual go to www invitrogen com protoarray and click on the Online Tools tab Install the Basic version of ProtoArray Prospector for data analysis The ProtoArray Prospector software currently accepts the output files GPR generated by the GenePix Pro microarray data acquisition software and analyzes the data using specified algorithms to generate a list of human proteins showing significant interactions with the protein probe 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 After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The proteins that score as positive in the experiment are proteins that satisfy the basic program options Based on the Z score and available protein sequence information we recommend validating the protein protein interaction by ProtoArray Technology or other methods as described on the next page Continued on next page Data Acquisition and Analysis Continued The Next Step Accessing Clones After identifying a positive interaction on the ProtoArray Human Protein or Yeast Proteome Microarray nc you may validate the protein
64. oarray nc is a high density protein microarray 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 This section provides details about the human protein microarray including array specifications and preparation of proteins Note The ProtoArray Human Protein Microarray PPI Complete Kits include 2 ProtoArray Human Protein Microarrays The specifications for the ProtoArray Human Protein Microarray nc are listed below Dimensions 1 inch x 3 inch 25 mm x 75 mm Material Glass slide coated with nitrocellulose membrane Membrane Size 20 mm x 60 mm Membrane Properties Thickness 15 20 um Pore Size 0 2 um Each microarray has a barcode for tracking samples The barcode is also used to retrieve array specific information from the ProtoArray Central Portal see page 42 The array specifications for the ProtoArray Human Protein Microarray nc are listed below The proteins on the microarray are printed in 48 subarrays and are equally spaced in vertical and horizontal directions For details on the subarray layout and human protein and control spots on the ProtoArray Human Protein Microarray nc go to the ProtoArray Central Portal at www invitrogen com protoarray Total Subarrays 48 4 columns x 12 rows Subarray Size 4400 um x 4400 um Subarray Dimensions 20 rows x 20
65. om This product is the subject of WIPO patent WO8809372 and foreign equivalents to be used for scientific investigation and research and for no other purpose whatsoever Licenses for commercial use of the above mentioned patents must be negotiated directly with Amrad Corporation 576 Swan Street Richmond Victoria Australia 3121 Phone 61 3 9208 4000 Continued on next page 57 Purchaser Notification Continued Limited Use Label License No 221 Microarrays of Biological Samples Limited Use Label License No 295 Polypeptides Expressed in Yeast 58 This product may be covered by one or more of U S Patent numbers 5 807 522 and 6 110 426 licensed exclusively to Incyte Corporation The purchase of this product conveys to the buyer whether employed in academia government not for profit entity or a for profit entity the limited non exclusive non transferable right without the right to resell repackage or further sublicense under these patents to use this product for research and development purposes No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially This product is sold pursuant to authorization from Incyte Corporation and Incyte Corporation reserves all other rights under these patents For information on
66. om the ProtoArray Central Portal see page 42 The array specifications for the ProtoArray Yeast Proteome Microarray are listed below The proteins on the microarray are printed in 48 subarrays and are equally spaced in vertical and horizontal directions For details on the subarray layout and yeast protein and control spots on the ProtoArray Yeast Proteome Microarray nc go to the ProtoArray Central Portal at www invitrogen com protoarray Note The subarray layout and controls are different in ProtoArray Yeast Proteome Microarray nc v1 1 as compared to the previously available ProtoArray Yeast Proteome Microarray nc v1 0 Total Subarrays 48 4 columns x 12 rows Subarray Size 4400 um x 4400 um Subarray Dimensions 16 rows x 20 columns Median Spot Diameter 150 um Spot Center to Center Spacing 220 um Distance Between Subarrays 100 um Replicates per Sample 2 Total Yeast Proteins on v1 1 array gt 4000 Refer to ProtoArray Central Portal for exact number of human proteins printed on the microarray Continued on next page ProtoArray Yeast Proteome Microarray Continued Preparing Yeast Proteins Printing the Yeast ProtoArray Controls Detecting Reciprocal Interactions Maintaining Stringent Quality Control 10 The yeast proteome collection is derived from the S cerevisiae clone collection of 5800 yeast ORFs Zhu et al 2001 Each S cerevisiae open reading frame ORF is
67. ormula on previous page to calculate the biotin reagent amount from each tube from Step 5 to the 3 protein sample tubes prepared as described on previous page See Fig C page 18 Add 1 35 ul Biotin Reagent 5 nmole ul from Step 5 tube 2 to the tube containing BSA Control Protein The total volume in each tube 22 ul protein x ul biotin reagent 7 Mix well by pipetting and centrifuge briefly in a microcentrifuge Avoid mixing the samples by vortexing 8 Incubate at room temperature for 60 minutes Proceed immediately to Purifying Biotinylated Protein next page Purifying Biotinylated Protein Introduction Materials Needed Experimental Outline Purification Module Preparing Spin Columns for Purification This section provides instructions to purify the biotin labeled protein from free biotin using the ProtoArray Biotinylation Purification Module The presence of free biotin in protein samples interferes with array probing and results in high background on the microarray e Biotinylated samples see previous page e ProtoArray Biotinylation Purification Module included in the ProtoArray complete kit Purify biotinylated protein probe using gel filtration N Estimate protein concentration of the purified biotinylated protein The ProtoArray Biotinylation Purification Module includes a gel filtration resin exclusion limit 6 000 Da and spin columns for rapid and efficient removal
68. ot reproducibility Pre Printing Quality Control Prior to production the arrayer and supporting components are tested and adjusted to production specifications To maintain protein stability and function arrays are printed at 6 C under controlled environmental conditions Post Printing Quality Control After production each microarray is visually inspected for obvious defects that could interfere with the experimental results To control for the quality of the printing process several microarrays from each lot are probed with an anti GST antibody Since the proteins contain a GST fusion tag probing the microarrays with an anti GST antibody allows identification of irregular spot morphology or missing spots The arrays are functionally qualified by probing control proteins to detect the appropriate protein protein interactions For detailed product qualification see page 55 Detecting ProtoArray Human Protein Microarrays are ideal for detecting reciprocal Reciprocal interactions since the microarrays are manufactured under highly controlled Interactions conditions to ensure maximum protein function Once you have identified a positive interaction using the ProtoArray Human Protein Microarray use the identified interacting protein from the array as a probe to probe another ProtoArray Human Protein Microarray nc to confirm the reciprocal interaction For example perform an initial probing with calmodulin as a probe with a Proto
69. otein the unconjugated or free biotin must be removed from the protein preparation as free biotin interferes with the probing procedure and increases the background on the array The ProtoArray Biotinylation Purification Module provides spin columns and purification resin to rapidly remove free biotin by gel filtration Since each protein is different the number of biotin molecules conjugated to the protein varies To prevent under biotinylation of the protein probe resulting in sub optimal sensitivity or over biotinylation of the protein probe resulting in loss of protein function it is important to verify and assess the biotin conjugation reaction The ProtoArray Biotinylation Assessment Module allows verification and assessment of the in vitro biotinylation reaction using Western detection The module includes a Biotinylation Gel Standard buffers and detection reagents to perform Western transfer and chemiluminescent detection using Streptavidin Alkaline Phosphatase AP conjugate The band intensity of the protein probe is compared to the Biotinylation Gel Standard to verify biotinylation and assess the level of biotinylation Based on the Western results you can choose the protein biotinylated at a suitable level to probe the array and minimize erroneous results due to the use of over or under biotinylated probe Continued on next page Description of Kit Components Continued ProtoArray PPI Buffer Module Alexa Fluor
70. ownload the latest version of the lot specific information files The GAL GenePix Array List files describe the location and identity of all spots on the Human Yeast and Control microarrays and are used with the microarray data acquisition software to generate files that contain pixel intensity information for feature spot and non features of the slide The GAL files are available for downloading from the ProtoArray Lot Specific Information available on ProtoArray Central see below Note The GAL files are text files that contain the data in a format specified by GenePix Pro Microarray data acquisition software If you are using any other microarray data acquisition software you can use data from the GAL files to generate files that are compatible with your microarray data acquisition software 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 as described below to portable media and then download the information onto the scanner computer 1 Go to www invitrogen com protoarray and then click on the Online Tools tab Click on the link to ProtoArray Lot Specific Information 3 Ent
71. protein interaction using the ProtoArray Technology or other methods Using the ProtoArray Technology validate the protein protein interactions by performing experiments with additional arrays to ensure e Reproducibility Probe the ProtoArray Human Protein or Yeast Proteome Microarray nc using a similar or a different probe concentration to observe similar interactions e Specificity Probe a ProtoArray Human Protein or Yeast Proteome Microarray nc with different biotinylated proteins to identify interactions specific to your protein probe of interest and also identify any non specific interactions e Reciprocal Interactions Determine reciprocal interactions using a purified protein probe see page 49 for an example Other methods for validating protein protein interactions include e Yeast Two Hybrid Systems page x e Co immunoprecipitation e Gel shift assay Since the majority of human proteins printed on the array are derived from the Ultimate ORF Clone Collection or purified proteins protein kinases available from Invitrogen itis very easy to order the clone or protein corresponding to the protein identified on the array and validate the interaction Visit www invitrogen com clones to access our clone collections Each Ultimate ORF Clone is full insert sequenced and guaranteed to match the corresponding GenBank amino acid sequence Contact Technical Support page 54 to order the purified protein kinases
72. purchasing a license for purposes other than research and development please contact Incyte Corporation Corporate Licensing Route 141 and Henry Clay Boulevard Wilmington DE 19880 Phone 302 498 6825 Fax 302 498 2707 This product is the subject of one or more of U S Patent Nos 5 618 676 5 854 018 5 856 123 5 919 651 and foreign equivalents Rights to use this product are limited to research use only Rights are available from Washington Research Foundation to practice under the above referenced patents for any use by contacting Washington Research Foundation 2815 Eastlake Avenue East Suite 300 Seattle Washington 98102 Tel 206 336 5600 Fax 206 336 5615 References Bouvier M Menard L Dennis M and Marullo S 1998 Expression and Recovery of Functional G protein coupled Receptors Using Baculovirus Expression Systems Curr Opin Biotechnol 9 522 527 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 Hollister J Grabenhorst E Nimtz M Conradt H and D L J 2002 Engineering the Protein N glycosylation Pathway in Insect Cells for Production of Biantennary Complex N glycans J Biochemistry 41 15093 15104 Kukar T Eckenrode S Gu Y Lian W Megginson M She J X and Wu D 2002 Protein Microarrays to Detect Protein Protein Interactions Using Red and Gree
73. r interactions with proteins Each human or S cerevisiae open reading frame ORF is expressed as an N terminal GST Glutathione S Transferase 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 protein assay performance since the nitrocellulose surface is known to be compatible with a variety of protein functions Espejo et al 2002 Kukar et al 2002 Michaud et al 2003 The nitrocellulose coating is thin and does not interfere with scanning of the array Each ProtoArray Protein Microarray PPI Complete Kit includes two microarrays to allow you to assay for protein interactions using different experimental conditions or two distinct proteins Using a labeled protein probe you can screen against the human or S cerevisiae proteins within a day to identify protein protein interactions For array specifications and more details on how the human and yeast proteins are prepared see pages 6 11 Continued on next page Description of Kit Components Continued ProtoArray Control Protein Microarray ProtoArray Mini Biotinylation and Purification Module ProtoArray Biotinylation Assessment Module The ProtoArray Control Protein Microarray nc contains human and yeast protein interactors and various controls printed on a nitrocellulose coated glass slide and is used to validate the biotinylation and pro
74. ray Microarray The amount of protein and quality of protein required for probing are described below After you have expressed your protein of interest follow the guidelines below to purify and prepare the protein probe e Purify the protein probe to gt 90 purity as determined by Coomassie staining e Resuspend the purified protein probe in a buffer lt 50 mM that does not contain any primary amines such as ammonium ions Tris glutathione imidazole or glycine If the buffer contains primary amines sufficiently dialyze proteins against 50 mM HEPES buffer pH 7 4 containing 100 mM NaCl or PBS e Know the approximate molecular weight of your protein Note The protein must be gt 15 kDa e For proteins purified using metal chelating column chromatography ProBond resin or Ni NTA resin perform dialysis against 2 changes of PBS to significantly lower the imidazole concentration e Ifyou are using a recombinant protein probe you may check the functionality of the protein using a method of choice e Low concentrations lt 0 1 of sodium azide or thimerosal in the protein solution have no effect on the biotinylation reaction You need at least 150 ug of purified protein at a concentration of 2 5 mg ml In Vitro Biotinylation Introduction ProtoArray Mini Biotinylation Module Biotin XX Sulfosuccinimidyl ester Sodium salt Biotin Protein Ratios Instructions are provided in this sect
75. ray Blocking Buffer 10X 10X PBS pH 7 4 1 Tween 20 ProtoArray Probe Buffer 5X 5X PBS pH 7 4 0 25 Triton X 100 25 Glycerol MgCl 1 M MgCl in deionized water TM HybriSlip Cover Slip 60 mm x 22 mm RNase free Array Chambers Continued on next page vil Kit Contents and Storage Continued ProtoArray Mini Biotinylation Module ProtoArray Biotinylation Purification Module viii The ProtoArray Mini Biotinylation Module includes the following reagents Store at 20 C Note Sufficient reagents are included to perform 4 in vitro biotinylation reactions Biotin XX sulfosuccinimidyl Lyophilized ester sodium salt Sterile water Control Protein BSA 2 5 ug l in PBS pH 7 4 Biotinylation Gel Standard 20 pmoles biotin conjugated per ml Biotinylated BSA of BSA in 1X NuPAGE LDS Sample Buffer 1X NuPAGE LDS Sample Proprietary Buffer 10X NuPAGE Reducing 500 mM stabilized dithiothreitol Agent DTT The ProtoArray Biotinylation Purification Module includes the following reagents Store Purification Resin at 4 C and Spin Columns and Collection Tubes at room temperature Note Sufficient reagents are included to perform 4 purifications Purification Resin 50 slurry in 50 mM HEPES pH 7 4 containing 0 1 M NaCl and 1 mM sodium azide Spin Columns with Collection Tubes Collection Tubes additional Continued on next page Kit Contents and Storage Conti
76. ring the probing procedure Avoid prolonged exposure of the Streptavidin Alexa Fluor 647 Conjugate to light Incorrect scanning or See above imaging High background Improper blocking Prepare the PBST Blocking Buffer fresh as described on page 32 Do not use the ProtoArray Blocking Buffer included in the kit without the addition of BSA Improper washing For the best results perform the recommended washing steps Prepare the Probing Buffer fresh as described on page 32 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array as described on page 35 before before scanning scanning Continued on next page 51 Troubleshooting Continued Control Array Results continued Uneven background Weak or no signal with biotinylated protein probe 52 Uneven blocking or washing Improper washing During the blocking or washing steps ensure the array is completely immersed in PBST Blocking solution or Probing Buffer and use at least 30 ml buffer in the Array Chamber to cover the array completely with buffer To obtain the best results perform the recommended washing steps Prepare the Probing Buffer fresh as described on page 32 Portions of array have dried Do not allow the array to dry during probing Improper array handling Biotinylated protein probe not applied properly Always wear gloves and avoid touching the surface of the array with
77. rotein sample to obtain the specified ratios of Y S ho biotin protein Add 1 35 ul of v d d y 5 nmoles ul biotin to the BSA Protein probe Protein probe BSA control Protein probe control sample Biotin Protein A 3 z Ratio ar 9 1 9 1 3 1 Continued on next page 18 In Vitro Biotinylation Continued Materials Needed Preparing Buffer Preparing Protein Samples Calculating the Amount of Biotin Reagent e Purified protein probe page 16 for amount and quality of protein e Biotin XX sulfosuccinimidyl ester sodium salt included in the ProtoArray Mini Biotinylation Module e Sterile water included in the ProtoArray Mini Biotinylation Module thaw and keep on ice until use e BSA Control Protein included in the ProtoArray Mini Biotinylation Module e Sterile microcentrifuge tubes e Freshly prepared 1 M sodium bicarbonate buffer see below for a recipe Prepare 1 M sodium bicarbonate fresh prior to use Dissolve 0 84 g of NaHCO in 10 ml deionized water The pH should be 8 3 8 5 Prepare protein samples for biotinylating at 3 molar ratios see Fig A previous page and BSA Control Protein included in the kit as follows 1 To 0 5 ml microcentrifuge tubes add the following on ice Components 27 1 9 1 3 1 BSA Protein Probe 2 5 mg ml 20 ul 20ul 20u1 BSA Control Protein 2 5 mg ml 20 ul 1 M Sodium Bicarbonate Buffer 2ul 2ul 2 ul 2 ul Mix well and
78. rotoArray Human Protein Microarray PPI Complete Kit v4 0 1 kit PAH0524013 for V5 epitope tagged proteins ProtoArray Yeast Proteome Microarray PPI Complete Kit v1 1 1 kit PA0121013 forV5 epitope tagged proteins ProtoArray Protein Protein Interaction Application Kit 1 kit PA013 for V5 epitope tagged proteins ProtoArray Mini Biotinylation Kit 1 kit AL 01 ProtoArray Human Protein Microarray KSI Complete Kit v4 0 1 kit PAH0524065 for kinase substrate identification ProtoArray Yeast Proteome Microarray KSI Complete Kit v1 1 1 kit PA0121065 for kinase substrate identification ProtoArray Kinase Substrate Identification Application Kit 1 kit PA015 ProtoArray Human Protein Microarray mg v4 0 1 array PAH052406 ProtoArray Yeast Proteome Microarray mg v1 1 1 array PA012106 ProtoArray Control Protein Microarray mg v4 0 1 array PA1002 ProtoArray Immune Response Biomarker Profiling 1 kit PA015 Application Kit ProQuest Two Hybrid System 1 kit PQ10002 01 ProQuest Two Hybrid System with Gateway Technology 1 kit PQ10001 01 Phosphate Buffered Saline PBS 1X 500 ml 10010 023 Pre Cast Gels and A variety of pre cast gels including NuPAGE Novex Pre cast Gels and pre Pre made Buffers made buffers for gel electrophoresis is available from Invitrogen For details on these products visit our website at www invitrogen com or contact Technical Support page 54 Overview Introduction ProtoArray Microarray PPI
79. should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose
80. store on ice until use Before Starting e Before starting the probing procedure make sure you have all items on hand especially buffers previous page biotinylated probes in Probing Buffer previous page Array Chambers included in the kit and HybriSlips included in the kit e Make sure the buffers are cold Store buffers on ice until use Place the Array Chambers on ice to chill the chamber until use e Review Important Guidelines on page 31 prior to starting the probing procedure Blocking Step Instructions for blocking the ProtoArray Control Protein Microarray nc are described below 1 Remove the mailer containing the ProtoArray Control Protein Microarray nc from storage at 20 C and immediately place the mailers at 4 C 2 Allow the mailer containing the array to equilibrate at 4 C for at least 15 minutes prior to performing the blocking step 3 Perform blocking in the mailer Ensure that the microarray is placed properly in the chamber with the printed white side facing up Add 30 ml PBST Blocking Buffer page 32 to the chamber containing the array Note You can block 2 arrays simultaneously in the mailer using 30 ml PBST Blocking Buffer Incubate for 1 hour in the cold room with gentle shaking 50 rpm 5 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 laborator
81. t of arrays Download the files listed above for human or yeast array specific information from a specific lot Use these files to interpret your results with the ProtoArray Human Protein or Yeast Proteome Microarray nc as described below Note The file size for some files such as the Protein Sequence File may be gt 1 MB Start the microarray data acquisition software on the computer and open the saved image tiff from Step 8 page 41 To acquire data from ProtoArray experiments e For GenePix Pro Software download the GAL files from ProtoArray Central for control or protein arrays which defines 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 control or 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 the proper location for each subarray Adjust the subarray grid if needed Continued on next page 43 Data Acquisition and Analysis Continued ProtoArray Central continued Analyzing Data Data Analysis Using ProtoArray Prospector ProtoArray Prospector Results 44 8 After the grid is properly adjusted and all features are aligned save export the results as a GPR GenePix Results file for data analysis using ProtoArray Prospec
82. te binds to the biotinylated anti mouse antibody e Cmdip signal The Array Control Protein BioEase V5 calmodulin kinase binds to the calmodulin printed on the array The signal is used to verify the probing procedure e V5 Control signal The V5 control protein contains an N terminal BioEase and V5 epitope tag and binds to the Streptavidin Alexa Fluor 647 conjugate Continued on next page Expected Results Continued Human The results obtained after probing the ProtoArray Human Protein Microarray ProtoArray nc v4 0 with 50 ug ml of the Array Control Protein i e BioEase V5 tagged Probing Results biotinylated calmodulin kinase probe is shown below Image showing Array Control Protein probe interacting with Cmd1p on the Human Microarray nc v4 0 Array Image Boxed Area shown in detail Alexa Fluor Ab AB Glomstp Alexa_ Alexa Fluor Biotin Ab Ab oo Continued on next page 47 Expected Results Continued Yeast ProtoArray The results obtained after probing the ProtoArray Yeast Proteome Microarray Probing Results ne v1 1 with 50 ug ml of the Array Control Protein i e BioEase V5 tagged biotinylated calmodulin kinase probe are shown below Image showing the Array Control Protein probe interacting with calmodulin Cmd1p on the array Array Image Boxed Area shown in detail Biotin Ab Alexa Fluor Ab Note The column of i
83. tinued Probes for Proteome Arrays Preparing Buffers Preparing Probes Before Starting The ProtoArray Human Protein or Yeast Proteome Microarray PPI Complete Kit contains 2 human or yeast arrays respectively and can be probed using the different probing options as described below Choose the option that best fits your needs The recommended protein probe concentration to use for probing the arrays is 5 50 ug ml Probing Options e You can probe both arrays simultaneously probing one array with your biotinylated protein probe and the second array with no protein probe negative control The negative control allows you to determine which signals are specific to your probe OR e You can probe one array with an initial probe concentration or biotinylation molar ratio If the initial signal is strong with low background confirm the initial results with the second array using the same experimental conditions If the initial results indicate weak signal and unacceptable signal to noise ratio probe the second array with a different probe concentration or molar ratio as described in the table below Probe first array And Then Probe Second Array With 5 ug ml probe Weak signal With 50 pg ml probe With 50 ug ml probe High background With 5 pg ml probe With 9 1 molar ratio Weak signal With 27 1 molar ratio With 27 1 molar ratio High background With 3 1 or 9 1 molar ratio Prepare
84. tor 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 After data acquisition analyze the data to identify protein interactions 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 This software allows rapid data analysis without the need to perform any manual calculations For more information see below Performing the data analysis by importing the data file into Microsoft Excel or an equivalent spreadsheet program to identify potential substrates is not recommended This approach requires a certain degree of expertise with statistics and Excel or another spreadsheet program The ProtoArray Prospector software quickly analyzes the data acquired from the image acquisition software and easily identifies statistically significant interactors 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 slides The Prot
85. uality of your in vitro biotinylated protein probe and the probing conditions you may proceed to probe the ProtoArray Human Protein or Yeast Proteome Microarray nc using your protein probe Follow the guidelines provided in this section ProtoArray Human Protein Microarray nc or ProtoArray Yeast Proteome Microarray nc included in the complete kit Note If you have purchased the ProtoArray Protein Protein Interaction Application Kit you also need to purchase the ProtoArray Human Protein or Yeast Proteome Microarray nc separately ProtoArray PPI Buffers Module A and B included in the kit Your biotinylated protein probe in Probing Buffer see next page Streptavidin Alexa Fluor 647 conjugate keep on ice in the dark until immediately before use Sterile 50 ml conical tube Ice bucket Deionized water Optional Microarray slide holder and centrifuge equipped with a plate holder Each ProtoArray Human Protein or Yeast Proteome Microarray nc can only be used once Do not re use the array or reprobe the same array with another probe ey 1 Block the ProtoArray Human Protein or Yeast Proteome Microarray nc Probe with your biotinylated protein probe Detect with Streptavidin Alexa Fluor 647 Conjugate Dry the array for scanning Follow the important guidelines on page 31 to obtain the best results with the arrays Continued on next page Probing the ProtoArray Human or Yeast Microarrays Con
86. ubate the arrays in the tube for 1 5 h at 2 8 C without shaking Remove each array from the conical tube and insert diagonally see Note below into an Array Chamber kept on ice Note The microarray with HybriSlip does not fit on the rails of the chamber You must insert the microarray diagonally into the chamber Using a sterile pipette add 25 ml Probing Buffer page 32 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 HybriSlip Using forceps carefully remove each HybriSlip without touching the array surface Do not remove the HybriSlip with the forceps if the HybriSlip is not dislodged from the array Discard the HybriSlip Reposition the array on the chamber rails if desired Incubate the array in Probing Buffer for 1 minute on ice Decant the Probing Buffer Invert chamber on paper towels for a few seconds to drain excess buffer Repeat Step 7 two more times using 25 ml Probing Buffer each time While the array is incubating prepare Streptavidin Alexa Fluor 647 Conjugate solution by mixing 6 ul of the Streptavidin Alexa Fluor 647 Conjugate included with the kit with 25 ml Probing Buffer After the third wash with Probing Buffer Step 8 decant the buffer Invert chamber on paper towels for a few seconds to drain excess buffer Add 25 ml of the Streptavidin Alexa Fluor 647 Conjugat
87. ure as analyzed by TLC and NMR analysis must indicate the correct structure Biotinylated Gel Standard BSA MALDI TOF Matrix Assisted Laser Desorption lonization Time Of Flight analysis must indicate the specified moles of biotin per mole of BSA The Purification Resin is qualified by loading 50 ug BSA solution and measuring the recovery The BSA recovery must be gt 35 ug The resin must also meet specifications for flow rate fractionation range and particle size Continued on next page 55 Product Qualification Continued ProtoArray The ProtoArray Biotinylation Assessment module is qualified as follows Biotinylation e The Western chemiluminescent detection reagents in the module are Sare qualified by performing electrophoresis of human IgG on a NuPAGE oquie Novex 4 12 Bis Tris Gel using NuPAGE MES SDS Running Buffer After electrophoresis protein is transferred onto a nitrocellulose membrane using NuPAGE Transfer Buffer Immunodetection is performed as described in this manual using anti human IgG primary antibody 10 100 pg antigen must be detected within 30 minutes of exposure The Streptavidin AP conjugate is tested in a Western blot with 12 5 fmoles of biotin conjugated to BSA A signal must be obtained using a 1 4000 dilution of Streptavidin AP and detection with a chemiluminescent substrate after a 30 second exposure to film Streptavidin Alexa The spectra for the Streptavidin Alexa Fluor 647
88. y wipe for a few seconds to drain any buffer without allowing the array to dry Place the array on a clean flat surface with the printed side of the array facing up 6 Proceed immediately to Probing the Array next page Continued on next page 33 Probing the ProtoArray Control Protein Microarrays Continued Probing Control Array 34 1 10 11 12 13 14 15 Pipet 120 ul of the biotinylated protein probe 50 ug ml in Probing Buffer page 32 on top of one control array without touching the array surface Add 120 pl Array Control Protein 50 ug ml in Probing Buffer on top of the second ProtoArray Control Protein Microarray nc The liquid quickly spreads over the nitrocellulose membrane Carefully lift the HybriSlip cover slip from the support liner with forceps and lay the clear side of HybriSlip on 1 array to cover the membrane area without trapping any air bubbles The HybriSlip is designed to exactly cover the membrane area Gently adjust the HybriSlip to remove any air bubbles Repeat for the second array Insert each assembly array with HybriSlip into a separate 50 ml conical tube with the printed side of the array facing up Cap the conical tube Place the conical tubes on a flat surface such that the printed side of the array is facing up and the tubes are as level as possible If needed you can tape the conical tubes on a flat surface to avoid any accidental disturbances Inc
89. ylated Protein from Step 3 page 28 An interaction of the biotinylated protein with the anti biotin antibody indicates that the protein is in fact biotinylated the biotins are accessible in solution and that the amount of free biotin remaining in the sample is low Use the biotinylated protein sample that gives the best signal on a Western blot at the lowest biotinylation molar ratio to probe the control array Array Control Protein Reacts with calmodulin printed on the Control Arrays use to verify probing procedure and reagents Continued on next page 31 Probing the ProtoArray Control Protein Microarrays Continued Array Control Protein Preparing Buffers 32 The Array Control Protein included in the complete kits is yeast calmodulin kinase Cmk1p containing an N terminal BioEase and V5 tag The presence of the BioEase tag facilitates in vivo biotinylation of the protein during expression see www invitrogen com for more information about BioEase vectors When probed against the ProtoArray Control Protein Microarray nc the biotinylated calmodulin kinase interacts with calmodulin Cmd1p printed on the array Detecting an interaction between the Array Control Protein and calmodulin indicates that the probing procedure has been performed correctly Prepare the following buffers fresh prior to use The recipe below provides sufficient buffer to probe 1 microarray To probe 2 microarrays scale up the a

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