RayBiotech, Inc.
Contents
1. Detection of auto antibodies The kit can be used to detect and characterize auto antibodies from body fluids RayBio is the trademark of RayBiotech Inc 5 6 RayBio Mouse Protein G Series Array Protocol Detection of protein modifications The kit can also be used to determine protein modifications such as phosphorylation Detection of protein DNA interaction In some cases the kit can be used to detect DNA binding proteins Features of RayBio Protein Arrays l High throughput approach allows simultaneous detection of multiple protein functions including protein protein interactions protein modifications antibody specificity presence of auto antibodies and small molecule protein interactions Affordable quick and simple to use Low sample consumption as little as 25 uL of original sample required per array Fully customizable create a custom array from our list of targets High sensitivity both biotin streptavidin pair and fluorescent detection enable the most sensitive assay High efficiency and accuracy high throughput screening of multiple targets in a single assay Each slide can test up to 2 samples simultaneously and contains internal positives to normalize between slides thereby minimizing the variation from assay to assay Additionally the assay duration is less than 6 hours Large dynamic range of detection 4 orders of magnitude with highly accurate data that can be normalized betw2. well Remove any bubbles on array surfaces Incubate arrays with gentle rocking or shaking at room temperature for 1 to 2 hours or at 4 C overnight or other condition as appropriate Note We recommend using 1 to 100 ug of total probe protein in your experiment If background is high use less amount of probe protein If the signals are weak use more protein Different protein protein interactions may need different binding buffers Note Blocking Buffer Item F can be used to dilute samples if necessary but PBS or other buffers may yield better results depending on the protein of interest 1 5 Dilute 20x Wash Buffer I Item G to 1x with ddH20 Decant the samples from each well and wash 5 times with 800 uL of 1x Wash Buffer I at room temperature with gentle shaking 5 minutes per wash Completely remove 1x Wash Buffer I in each wash step as recommended in section Handling Glass Slide Chips Part B Note Avoid solution flowing into neighboring wells Il RayBio Mouse Protein G Series Array Protocol 1 6 Dilute 20x Wash Buffer II Item H to 1x with ddH20 Wash 2 times with 800 uL of 1x Wash Buffer II at room temperature with gentle shaking 5 minutes per wash Completely remove 1x Wash Buffer II in each wash step Detection of associated protein Depending on the different strategies in the experimental design different protocols can be used gt If a biotin labeled protein sample is used as the probe Step 1
3. 4 above Figure 1 bottom panel follow the procedures described below 2 1 Briefly spin the vial containing 1 500x Fluorescence conjugated Streptavidin Item E prior to use add 1 5 mL of Blocking Buffer Item F and mix well Spin the vial briefly and add 400 uL of diluted Fluorescence conjugated Streptavidin to each sub array 2 2 Cover the incubation chamber with adhesive strips Item I Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 2 3 Incubate at room temperature for 1 to 2 hours with gentle rocking or shaking Note Incubation may be done at 4 C overnight 2 4 Wash with 1x Wash Buffer I as described in Step 1 5 and 1x Wash Buffer II as described in Step 1 6 above Continue on Step 3 1 gt Ifanon labeled protein sample is used as the probe Step 1 4 above Figure l top and center panels follow the 12 RayBio Mouse Protein G Series Array Protocol procedures described below However the following assay requires an antibody against the probe protein or its fused RayBio Mouse Protein G Series Array Protocol down Add 400 uL of diluted Fluorescence conjugated Streptavidin to each sub array tag s User will need to purchase or create these antibodies as they are not provided in the kit 2 7 Cover the incubation chamber with adhesive strips Item D Cover the plate with aluminum foil to avoid exposure to light 2 1 Add 400 uL of appropriately diluted antibody agai
4. RayBio Mouse Protein Array G Series Glass chip based protein arrays User Manual Revised December 04 2015 For detecting protein protein interactions antibody specificity auto antibodies protein modifications and small molecule protein interactions RayBio Mouse Protein Array G Series Cat PAM G2 RayBio Custom Mouse Protein Array G Series Cat PAM CUST G RayBio Mouse Protein Array G Series Service Cat PAM SERV G RayBiotech Inc We Provide You with Excellent Protein Array Systems and Service Tel 1 888 494 8555 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com E mail info raybiotech com RayBio Mouse Protein G Series Array Protocol RayBio Mouse Protein Array Target List Please visit our website http Avwww raybiotech com to download the list of targets printed on glass slides RayBio Mouse Protein Array Map Template Please visit our website http Awww raybiotech com to download the map template Additional Custom Protein Array Services We Provide We also offer the completely customized protein arrays with many options that can be requested by a customer We can help with experimental design in selecting the most appropriate array for your needs designing the experiment to detect your sample of need or just help with technical questions For more information please contact us 1 Experiment Design RayBiotech s protein array experts can assist you in your e
5. V Protocols A Detection of Protein Protein Interactions Several strategies can be used for detection of protein protein interaction as shown in Figure 1 55 22 Fusion protein A Anti tag Aitietag antibody E Ca ow Figure 1 Three common strategies for detection of protein protein interactions using RayBio Protein Array kits 1 Blocking and Sample Incubation 1 1 Take the package containing the Assembled Glass Slide Item A from the freezer Place unopened package on the bench top for approx 15 30 minutes and allow the Assembled Glass Slide to equilibrate to room temperature 1 2 Open the package and take the Assembled Glass Slide out of the sleeve Do not disassemble the Glass Slide from the chamber assembly Place glass slide assembly in laminar flow hood or similar clean environment for 1 2 hours at room temperature 10 RayBio Mouse Protein G Series Array Protocol Note Protect the slide from dust or others contaminants 1 3 Block sub arrays by adding 400 uL of Blocking Buffer Item F into each well of Assembled Glass Slide Item A and incubating at room temperature for 30 minutes Ensure there are no bubbles on the array surfaces Note Only add reagents to wells printed with proteins Do not forcefully pipette any buffers samples etc onto the arrays Slowly pipette these reagents down the sides of the well 1 4 Decant the Blocking Buffer from each well Add 400 pL protein probe to each
6. array s e Xs the signal density for a particular spot X on sample array s e nXs the normalized Xs value Caution for interpretation of results 1 The in vitro and in vivo protein function may behave differentially Some recombinant proteins contain a tag sequence Some recombinant proteins on the array lack certain domains of the total protein particularly hydrophobic domains The folding status of those proteins is largely unknown All these may affect protein protein interactions 2 Almost all membrane proteins arrayed on glass slides contain extracellular and cytoplasmic domains but lack transmembrane domains 3 Different proteins may require distinct conditions for their optimal function recognition or antibody binding Therefore investigators in some cases may need to use different conditions for array testing 4 Always perform control experiments since both IgG and streptavidin may bind to some proteins 27 RayBio Mouse Protein G Series Array Protocol VI Troubleshooting Guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Ensure sufficient incubation time or change Weak Signal Short incubation times sample incubation to an overnight step Protein or antibody concentrations Dilute starting sample less or concentrate in sample are too low sample Store ki
7. aterials Required 7 our available list or provide your own proteins and RayBiotech LiL Overview and General Considerations Inc then produces your custom protein arrays for you A Preparation of Samples 7 Applications B Handling Glass Chips 8 Since RayBio protein arrays have multiple applications which C Incubation 8 a require different procedures only some examples of the potential D Layout of Mouse Glass Chips 9 uses of our protein array are given here E Incubation Chamber Assembly 9 a 1 Detection of protein protein interactions The kit can be IV Protocol used to screen novel protein protein interactions validate the A Detection of Protein Protein Interactions 10 previously known protein protein interactions and S n investigate the molecule interaction conditions B Characterization of Antibody Specificity 15 f l DO 2 Characterization of antibodies The kit can be used to test C Detection of Auto antibodies 20 the specificity of an antibody for research and therapeutic D Detection of Small Molecule protein Interaction 24 antibody development and find out the potential cross tion to oth teins E Detection of Protein Modifications 25 Retr ene renee vere V Data E d Analysi 26 3 Target identification The kit can be used to screen the aia Patmacwon Hind anaes small molecule protein interaction for target identification VI Troubleshooting Guide 28 drug discovery and toxicity study VII Reference List 29 4
8. e sleeve Do not disassemble the Glass Slide from the chamber assembly Place glass slide assembly in laminar flow hood or similar clean environment for 1 2 hours at room temperature Note Protect the slide from dust or others contaminants 16 RayBio Mouse Protein G Series Array Protocol 1 3 Block sub arrays by adding 400 uL of Blocking Buffer Item F into each well of Assembled Glass Slide Item A and incubating at room temperature for 30 minutes with gentle shaking Ensure there are no bubbles on the array surfaces Note Only add reagents to wells printed with proteins 1 4 Decant Blocking Buffer from each well Add 400 uL of test antibody sample to each well Remove any bubbles from the array surfaces Incubate arrays with gentle rocking or shaking at room temperature for 1 to 2 hours overnight at 4 C or other condition as appropriate Note Blocking Buffer Item F can be used to dilute samples if necessary 1 5 Dilute 20x Wash Buffer I Item G to 1x with ddH2O Decant the samples from each well and wash 5 times with 800 uL of 1x Wash Buffer I at room temperature with gentle shaking 5 minutes per wash Completely remove 1x Wash Buffer I in each wash step Note Avoid solution flowing into neighboring wells 1 6 Dilute 20x Wash Buffer II Item H to 1x with ddH20 Wash 2 times with 800 uL of 1x Wash Buffer II at room temperature with shaking 5 minutes per wash Completely remove 1x Wash Buffer II in each wash
9. ePix using the cy3 green channel Note Although we recommend scanning slides right after experiment you also can store the slide at room temperature or 20 C in dark place for several days Cy3 fluors dye used in this kit is very stable at room temperature and resistant to photo bleaching on completed glass slides If you do not have a laser scanner please send your slides to us and we can scan them for you for free B Characterization of Antibody Specificity Several strategies can be used for detection of antibody specificity as shown below in Figure 2 If needed RayBiotech can assist you in your experiment design and provide testing services for your project Please feel free to contact us with your questions so that we can assist in your project 15 RayBio Mouse Protein G Series Array Protocol Aa fer sy Labeled Nes 2 Ab Ds Eg ss tanto a Figure 2 Determination of interest antibody specificity using RayBio Protein Array kits The following protocol is for use with the biotin conjugated anti human mouse or rabbit IgG secondary antibody provided in this kit Items B C and D 1 Blocking and Sample Incubation 1 1 Take the package containing the Assembled Glass Slide Item A from the freezer Place unopened package on the bench top for approx 15 30 minutes and allow the Assembled Glass Slide to equilibrate to room temperature 1 2 Open package and take the Assembled Glass Slide out of th
10. ed glass slides If you do not have a 19 RayBio Mouse Protein G Series Array Protocol laser scanner please send your slides to us and we can scan them for you for free C Detection of Auto antibodies The following strategy can be used for detection of mouse auto antibody as shown in the following Figure 3 RayBiotech can assist you in your experimental design and provide testing services for your project Please feel free to contact us with any questions Figure 3 Detection of auto antibodies using RayBio Protein Array kits 1 Blocking and Sample Incubation 1 1 Take the package containing the Assembled Glass Slide Item A from the freezer Place unopened package on the bench top for approx 15 30 minutes and allow the Assembled Glass Slide to equilibrate to room temperature 1 2 Open package and take the Assembled Glass Slide out of the sleeve Do not disassemble the Glass Slide from the chamber assembly Place glass slide assembly in laminar flow hood or similar clean environment for 1 2 hours at room temperature 20 RayBio Mouse Protein G Series Array Protocol Note Protect the slide from dust or others contaminants 1 3 Block sub arrays by adding 400 uL of Blocking Buffer Item F into each well of Assembled Glass Slide Item A and incubating at room temperature for 30 minutes Ensure there are no bubbles on the array surfaces Note only add reagents to wells printed with proteins 1 4 Decan
11. een arrays II Materials Provided RayBio Mouse Protein G Series Array Protocol Storage Upon receipt all components in the kit should be stored at 20 C to 80 C until just before the experiment If stored at 20 C to 80 C the kit will retain complete activity for up to 6 months Please use within 6 months of purchase Once thawed protein array glass slide Item A and Blocking Buffer Item F should be kept at 20 C and all other components Items B E G amp H should be stored at 4 C Use within 3 months after reagents have been thawed Kit Components Item Description Cat PAM G2 2 Cat PAM G2 4 Cat PAM G2 8 RayBio H Protein A A ave oe eee 1 slide 2 slides 4 slides Glass Slides B 1 000 X Biotin conjugated Anti Lvial 2 Vials 3 vials Mouse IgG 1 5 ul vial 1 000 X Biotin conjugated Anti i i C 4 g 1 vial 2 vials 3 vials Rabbit IgG 1 5 ul vial 1 000 X Biotin conjugated Anti D Sum cannes Rete 1 vial 2 vials 3 vials Human IgG 1 5 ul vial 1 500 x HiLyte 555 Streptavidi E totes yte preven 1 vial 2 vials 3 vials Fluor 1 pl vial F Blocking Butter 1 bottle 1 bottle 2 bottles 8 ml bottle G 20X Wash Buffer 1 bottle 1 bottle 2 bottles 30 ml bottle H 20 Wash Butter it 1 bottle 1 bottle 2 bottles 30 ml bottle Adhesive Plastic Strips 1 strip 2 strips 4 strips J 30 ml Centrifuge Tube 1 tube 1 tube 1 tube K User Manual Plea
12. eveal used for clinical diagnostics Our products may not be resold ubiquilin 1 as a humoral immune response target in lung modified for resale or used to manufacture commercial products adenocarcinoma Cancer Res 67 3461 3467 without written approval by Raybiotech Inc e Huang R P 2003a Cytokine antibody arrays a promising Under no circumstances shall RayBiotech be liable for any tool to identify molecular targets for drug discovery Comb damages arising out of the use of the materials Chem High Throughput Screen 6 769 775 Products are guaranteed for three months from the date of purchase e Huang R P 2003b Protein arrays an excellent tool in when handled and stored properly In the event of any defect in biomedical research Front Biosci 8 D559 D576 quality or merchantability RayBiotech s liability to BUYER for any claim relating to products shall be limited to replacement or e Zhu H Bilgin M Bangham R Hall D Casamayor A refund of the purchase price 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 This product is for research use only 2015 RayBiotech Inc 29 30
13. iotin labeled Anti Mouse IgG Item B Add 1 5 mL of Blocking Buffer Item F and mix well 1 8 Incubate at room temperature for 1 hour 1 9 Wash with 1x Wash Buffer I as described in Step 1 5 and 1x Wash Buffer II as described in Step 1 6 above 1 10 Briefly spin the vial containing 1 500x Fluorescence conjugated Streptavidin Item E prior to use add 1 5 mL of Blocking Buffer Item F and mix well Add 400 uL of diluted Fluorescence conjugated Streptavidin to each sub array 1 11 Cover the incubation chamber with adhesive strips Item D Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 1 12 Incubate at room temperature for 1 hour with gentle rocking or shaking 1 13 Wash with 1x Wash Buffer I as described in Step 1 5 above and 1x Wash Buffer II as described in Step 1 6 above 2 Fluorescence Detection 2 1 Decant excess 1x Wash Buffer II from wells 2 2 Carefully disassemble the glass slide from the incubation frame and chamber by pushing clips outward from the sides 22 RayBio Mouse Protein G Series Array Protocol as shown below Carefully remove the glass slide from the gasket Note Be careful not to touch the printed surface of the glass slide which is on the same side as the barcode 2 3 Place the whole slide in a 30 mL Centrifuge Tube tem J Add enough 1x Wash Buffer I about 30 mL to cover the whole slide and gently shake or rock at room temperature for 10
14. minutes Decant 1x Wash Buffer I Repeat wash step with 1x Wash Buffer I once 2 4 Wash with 1x Wash Buffer II about 30 mL with gentle shaking at room temperature for 10 minutes Decant 1x Wash Buffer II 2 5 Rinse the glass slide with 30 mL of ddH2O for 5 minutes Remove glass slide and decant water from 30 mL Centrifuge Tube 2 6 Remove excess liquid from 30 mL Centrifuge Tube and place glass slide back into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets on glass slide and then let slide air dry completely at least 20 minutes protect from light Note Make sure the slides are absolutely dry before starting the scanning procedure or storage 2 7 Capture the signals using laser scanner such as Axon GenePix using cy3 green channel 23 RayBio Mouse Protein G Series Array Protocol Note Although we recommend scanning slides right after experiment you also can store the slide at room temperature or 20 C in dark for several days Cy3 fluors dye used in this kit is very stable at room temperature and resistant to photo bleaching on completed glass slides If you do not have a laser scanner send your slides to us and we can scan them for you D Detection of Small Molecule Protein Interaction Several strategies can be used for detection of small molecule protein interaction as outlined and suggested in Figure 4 RayBiotech can assist you in your experiment design and provide service for your p
15. nst the or incubate in dark room probe protein or its fused tag s into each well Incubate at room temperature for 2 hours 2 8 Incubate at room temperature for 1 hour with gentle rocking or shaking Note Incubation may be done at 4 C for overnight Usually 1 ng mL to 1 000 ng mL of antibody will be used You will need to optimize the dilution factor for your particular antibody in this experiment Blocking Buffer Item F can be used for dilution 2 9 Wash with 1x Wash Buffer I as described in Step 1 5 and 1x Wash Buffer II as described in Step 1 6 above 3 Fluorescence Detection 2 2 Wash with 1x Wash Buffer I as described in Step 1 5 above and 1x Wash Buffer II as described in Step 1 6 above 3 1 Decant excess 1x Wash Buffer II from wells 2 3 Add 400 uL of 1 000 fold diluted Biotin labeled antibody to 3 2 Carefully disassemble the glass slide from the incubation each well The choice of biotinylated secondary antibody will depend on the antibody chosen for protein recognition For example choose Biotin labeled Anti Mouse IgG tem B if the probe antibody derives from mouse choose Biotin labeled Anti Rabbit IgG Item C if the probe antibody derives from rabbit etc To prepare 1 000 fold diluted Biotin labeled Anti IgG spin down the vial containing Biotin labeled Anti IgG briefly add 1 5 mL of Blocking Buffer Item F and mix well frame and chamber by pushing clips outward from the sides as shown below Carefully remo
16. ody specific for your protein To profile auto antibodies you need to prepare your serum or plasma Optimal sample dilutions and amounts will need to be determined by each experimenter empirically Blocking Buffer Item F can be used to dilute samples if necessary but PBS or other buffers may yield better results depending on the protein of interest Normalize samples by loading equal amounts or equal dilutions Optimization of experimental conditions If you experience high background you may need to further dilute your sample and or to wash slides in Wash Buffer I overnight at 4 C If the signal is too weak you may need to increase the amount of your sample and or increase incubation times of one or more steps Completely cover array area with sample or buffer during incubation steps Cover the incubation chamber with adhesive strips Item I during incubation or plastic sheet protector to avoid drying particularly when incubation lasts more than 2 hours or less than 400 uL of sample or reagent is used During incubation and wash steps avoid foaming and remove any bubbles from the sub array surface Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle second Avoid cross contamination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Slide Assembly to decant and aspirate the remaining liquid see picture above Seve
17. ral incubation steps such as blocking sample incubation biotin conjugated antibody incubation or fluorescence conjugated streptavidin incubation may be done at 4 C overnight Before overnight incubations cover the incubation chamber tightly to prevent evaporation Protect glass slides from direct strong light and temperatures above room temperature RayBio Mouse Protein G Series Array Protocol D Layout of Mouse Glass Arrays e Each slide contains identical sub arrays see picture right e Don t touch the printed surface of the glass slide which is on the same side as the barcode MILLI LAN 123456789 Sub array Sub array E Incubation Chamber Assembly After finishing your experiment and disassembling the incubation chamber if you need to repeat any of the incubation or wash steps you must first re assemble the glass slide into the incubation chamber by following the steps as shown in the figures below To avoid breaking the printed glass slide it is recommended that you first practice assembling the device with a standard glass histology or microscope slide e Apply slide to incubation chamber barcode facing upward Image A e Gently snap one edge of a snap on side Image B e Gently press other of side against lab bench and push in lengthwise direction Image C e Repeat with the other side mage D A Gs ve LA RayBio Mouse Protein G Series Array Protocol I
18. roject Please contact us with questions or suggestion on experimental design a say Q 5 s ae os Protein linked molecule Figure 4 Detection of small molecule protein interaction using RayBio Protein Array kit 24 RayBio Mouse Protein G Series Array Protocol E Detection of Protein Modifications RayBio Mouse Protein Arrays may also be used to detect protein modifications such as protein phosphorylation modifications Figure 5 aia inact a phosphorylated Ab Figure 5 Detection of mouse protein phosphorylation modifications using RayBio Protein Array kit RayBiotech can assist you in your experiment design and provide service for your project Please contact us with your questions or for suggestions on experimental design 23 RayBio Mouse Protein G Series Array Protocol V Data Extraction and Analysis The captured array signal can be extracted with most of the microarray analysis softwares GenePix ScanArray Express ArrayVision etc associated with the laser scanner The signal intensities obtained from laser scanner can simply be analyzed by importing the fluorescence values into our analysis tool Cat 02 PAM G2 RayBiotech supports each array kit by offering Excel based analysis software tools for the automatic computation of the extracted numerical data obtained from the array image Features include sorting averaging background subtraction positive control normaliza
19. se download online www raybiotech com L Array Target List Please download online www raybiotech com M Array Map Template Please download online www raybiotech com Notes Items B E dilute with Blocking Buffer Item F prior to use Items G amp H dilute with ddH 0 prior to use RayBio Mouse Protein G Series Array Protocol B Handling Glass Chips RayBio Mouse Protein G Series Array Protocol Additional Materials Required Depending on your specific purpose different additional materials may be needed such as e The microarray slides are delicate Do not touch the array surface with pipette tips forceps or your fingers Hold the Small plastic boxes or containers slides by the edges only Pipettors pipette tips and other common lab consumables Handle the slides with powder free gloves and in a clean Orbital shaker or oscillating rocker environment Aluminum foil Remove reagents sample by gently ddH gt O applying suction with a pipette to corners of each chamber see picture right Do not touch the printed area of the array only the sides Laser scanner for fluorescence detection III Overview and General Considerations C Incubation A Preparation of Samples Depending on your experimental purpose different sample types may be used To detect protein protein interaction you may use your protein of interest as a probe either by labeling your protein with biotin or another reporter or by using an antib
20. shown below Carefully remove the glass slide from the gasket 18 RayBio Mouse Protein G Series Array Protocol Note Be careful not to touch the printed surface of the glass slide which is on the same side as the barcode 2 3 Place the whole slide in a 30 mL Centrifuge Tube tem J Add enough 1x Wash Buffer I about 30 mL to cover the whole slide and gently shake or rock at room temperature for 10 minutes Decant 1x Wash Buffer I Repeat wash step with 1x Wash Buffer I once 2 4 Wash with 1x Wash Buffer II about 30 mL with gentle shaking at room temperature for 10 minutes Decant 1x Wash Buffer II 2 5 Rinse the glass slide with 30 mL of ddH2O for 5 minutes Remove glass slide and decant water from 30 mL Centrifuge Tube 2 6 Remove excess liquid from 30 mL Centrifuge Tube and place glass slide back into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets on glass slide and then let slide air dry completely at least 20 minutes protect from light Note Make sure the slides are absolutely dry before starting the scanning procedure or storage 2 7 Capture the signals using laser scanner such as Axon GenePix using cy3 green channel Note Although we recommend scanning slides right after experiment you also can store the slide at room temperature or 20 C in dark for several days Cy3 fluors dye used in this kit is very stable at room temperature and resistant to photo bleaching on complet
21. step 1 7 Add 400 uL of 1 000 fold diluted appropriate Biotin labeled secondary antibody For example choose Biotin labeled Anti Mouse IgG Item B if the probe antibody derives from mouse choose Biotin labeled Anti Rabbit IgG Item C if the probe antibody derives from rabbit etc To prepare 1 000 fold diluted Biotin labeled Anti IgG briefly spin down the 17 RayBio Mouse Protein G Series Array Protocol vial containing Biotin labeled Anti gG add 1 5 mL of Blocking Buffer Item F and mix well 1 8 Incubate at room temperature for 1 hour 1 9 Wash with 1x Wash Buffer I as described in Step 1 5 and 1x Wash Buffer II as described in Step 1 6 above 1 10Briefly spin the vial containing 1 500x Fluorescence conjugated Streptavidin Item E prior to use Add 1 5 mL of Blocking Buffer Item F and mix well Add 400 uL of diluted Fluorescence conjugated Streptavidin to each sub array 1 11 Cover the incubation chamber with adhesive strips Item D Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 1 12 Incubate at room temperature for 1 hour with gentle rocking or shaking 1 13 Wash with 1x Wash Buffer I as described in Step 1 5 above and 1x Wash Buffer II as described in Step 1 6 above 2 Fluorescence Detection 2 1 Decant excess 1x Wash Buffer II from wells 2 2 Carefully disassemble the glass slide from the incubation frame and chamber by pushing clips outward from the sides as
22. t Blocking Buffer from each well Add 400 uL of appropriately diluted mouse serum plasma or other sample fluids to each well Remove any bubbles on array surfaces Incubate arrays with gentle rocking or shaking at room temperature for 1 to 2 hours or overnight at 4 C or other condition as appropriate Suggested dilution of serum or plasma is 10 to 200 fold Note Since auto antibody concentrations in mouse serum and plasma may vary widely you may need to optimize this dilution for your samples We usually use 100 fold dilution in our own experiments Note Blocking Buffer Item F can be used to dilute samples if necessary but PBS or other buffers may yield better results 1 5 Dilute 20x Wash Buffer I Item G to 1x with ddH20 Decant the samples from each well and wash 5 times with 800 uL of 1x Wash Buffer I at room temperature with gentle shaking 5 minutes per wash Completely remove 1x Wash Buffer I in each wash step Note Avoid solution flowing into neighboring wells 1 6 Dilute 20x Wash Buffer II Item H to 1x with ddH20 Wash 2 times with 800 uL of 1x Wash Buffer II at room 21 RayBio Mouse Protein G Series Array Protocol temperature with gentle shaking 5 minutes per wash Completely remove 1x Wash Buffer II in each wash step 1 7 Add 400 uL of 1 000 fold diluted Biotin labeled Anti Mouse IgG Item B to each well To prepare 1 000 fold diluted Biotin labeled Anti Mouse IgG briefly spin down the vial containing B
23. t as suggested temperature Don t freeze thaw the slide xcess of protein or antibody Further dilute protein or antibody Excess of streptavidin Further dilute streptavidin Overexposure Lower the laser power Improper storage of kit Minimize dust in work environment before Dust gt starting experiment High Background Handle and pipette solutions more gently De Bubbles formed during incubation gas solutions prior to use Cover the incubation chamber with adhesive Uneven Signal Reagent evaporation film during incubation Arrays are not completely covered Prepare more reagent and completely cover by reagent arrays with solution Slide is allowed to dry out Take additional precautions to prevent slides from dying out during experiment Dark Spots Completely remove wash buffer in each wash step Insufficient wash Increase wash time and use more wash buffer Feel free to call us if your question doesn t match this table 28 RayBio Mouse Protein G Series Array Protocol RayBio Mouse Protein G Series Array Protocol VII Reference List e Chen G Wang X Yu J Varambally S Yu J Thomas D G i Lin M Y Vishnu P Wang Z Wang R Fielhauer J RayBio is the trademark of RayBiotech Inc Ghosh D Giordano T J Giacherio D Chang A C Orringer M B El Hefnawy T Bigbee W L Beer D G and This product is intended for research purposes only and is not to be Chinnaiyan A M 2007 Auto antibody profiles r
24. tion and histogram graphing for easy visual comparison This analysis tool is very simple and affordable which will not only assist in compiling and organizing your data but also reduces your calculations to a copy and paste step Data normalization The raw data normalization is used to compare data between arrays i e different samples by accounting for the differences in signal intensities of the positive control spots on those arrays The positive control is a controlled amount of biotinylated protein printed on the arrays in triplicate The amount of signal from each of those spots is dependent on the amount of the reporter Cy3 streptavidin bound to biotinylated protein Since the reporter amount proportionally affects the signal intensity of every spot on the array the differences in the positive control signals between arrays will accurately reflect the differences between other spots on those arrays To normalize the data one array must be defined as the Reference Array r to which the signals of other Sample Arrays s are 26 RayBio Mouse Protein G Series Array Protocol normalized It is up to the customers to define which array should be the reference The normalized values are calculated as follows Pr Ps nXs Xs x e Pr the average signal density of the positive control spots on the reference array r e Ps the average signal density of the positive control spots on the sample
25. ve the glass slide from the gasket Note Be careful not to touch the printed surface of the glass slide which is on the same side as the barcode 2 4 Incubate at room temperature for 1 hour 3 3 Place the whole slide in a 30 mL Centrifuge Tube tem J Add enough 1x Wash Buffer I about 30 mL to cover the whole slide and gently shake or rock at room temperature for 10 minutes Decant 1x Wash Buffer I Repeat washing step with 1x Wash Buffer I once 2 5 Wash with 1x Wash Buffer I as described in Step 1 5 and 1x Wash Buffer II as described in Step 1 6 above 2 6 Briefly spin down the vial containing 1 500x Fluorescence conjugated Streptavidin Item E prior to use and add 1 5 mL of Blocking Buffer Item F and mix well Briefly spin vial 13 14 RayBio Mouse Protein G Series Array Protocol 3 4 Wash with 1x Wash Buffer II about 30 mL with gentle shaking at room temperature for 10 minutes 3 5 Rinse the glass slide with 30 mL of ddH O for 5 minutes Remove glass slide and decant water from 30 mL Centrifuge Tube 3 6 Remove excess liquid from 30 mL Centrifuge Tube and place glass slide back into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets on glass slide and then let slide air dry completely at least 20 minutes protect from light Note Make sure the slides are absolutely dry before starting the scanning procedure or storage 3 7 Capture the signals using laser scanner such as Axon Gen
26. xperiment design based on your project purpose 2 Customized Arrays e Select your targets from our RayBio Mouse Protein Array lists e Send your targets to us such as proteins synthesized polypeptides DNA lipids and any other molecules elf your targets are not available on the market we can produce recombinant proteins using our rapid bacterial gene expression system or mammalian cell gene expression system For small polypeptides we also provide peptide synthesis service 3 Full Testing Services You can send your samples to us and our expert staff will run the experiments and provide you with the fully analyzed results RayBio Mouse Protein G Series Array Protocol RayBio Mouse Protein G Series Array Protocol I Introduction RayBio Protein Arrays are a series of products developed by Protocol for RayBiotech Inc The Protein Array Pioneer Company Native or RayBio Mouse Protein Array G Series recombinant proteins are spotted onto the surface of a solid glass slide support The kits can be applied in screening protein protein interactions monitoring the presence of auto antibodies determining antibody specificity identifying protein modifications and or detecting small molecule protein interactions TABLE OF CONTENTS I Introduction Il Materials Provided 6 Fully customizable protein arrays are also available from RayBiotech Inc You can select your own proteins of interest from Additional M
Download Pdf Manuals
Related Search