User Manual
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1. 51 GlIcNAc B 1 4 GlcNAc B 2 B Gal 52 B D GlcA 3 a Man 53 Gal B 1 4 6S GINAc B GIcNAc a 1 3 Glc a 1 2 Glc a 1 2 Gal 4 a Fuc 54 a 1 3 Glc a Gal B 1 3 GalNAc B 1 4 Neu5Gc a 2 3 5 a Rha 55 Gal B 1 4 Glc B 6 B GleNAc 56 Sisomicin Sulfate GalNAc a 1 3 Fuc a 1 2 Gal B Blood 7 B GalNAc 57 A antigen trisaccharide Fuc a 1 2 Gal B 1 4 GlcNAc Blood H 8 Tobramycin 58 O f antigen trisaccharide Gal a 1 3 Fuc a 1 2 Gal B Blood B 9 Gal B 1 3 GIcNAc B 59 i RayBio Glycan Array 100 antigen trisaccharide 16 10 Gal a 1 3 Gal B 1 3 GIcNAc B 11 Neu5Ac a 2 3 Gal B 1 3 GIlcNAc B 12 NeuS5Ac a 2 6 Gal 1 3 GlcNAc 13 Neu5Gc a 2 3 Gal B 1 3 GIlcNAc B 14 Neu5Gc a 2 6 Gal B 1 3 GIlcNAc B Gal B 1 3 Fuc a 1 4 GIcNAc B Lewis A 16 Gal B 1 4 Glc B 17 Gal a 1 3 Gal B 1 4 Glc B 18 Gal a 1 4 Gal B 1 4 Glc B 15 19 GIcNAc B 1 3 Gal B 1 4 Glc B 20 GalNAc B 1 3 Gal B 1 4 Glc B 21 NeuSAc a 2 3 Gal B 1 4 Glc B 22 NeuSAc a 2 6 Gal B 1 4 Glc B 23 Neu5Gc a 2 3 Gal 1 4 Glc 24 NeuSAc a 2 6 Gal B 1 4 Glc B 25 Gal B 1 4 Fuc a 1 3 Glc B 26 GalNAc B 1 3 Gal a 1 4 Gal B 1 4 Glc B 27 GIcNAc B 1 6 GIcNAc B 28 4 P GIcNAc B 1 6 GIcNAc B 29 Glc a 1 2 Gal a 1 3 Glc a 30 Gal 1 3 GalNAc a 31 Gal 1 4 GIcNAc P Gal B 1 4 Fuc a 1 3 GlcNAc B Lewis x NeuSAc a 2 3 Gal 1 4 Fuc a 1 3 GIcNAc Sialyl Lewis X NeuSAc a 2 3 Gal 1 3 Fuc a 1 4 GIcNAc2. Introduction Glycocalyx literally meaning sugar coat is an extracellular polymeric coating surrounding many prokaryotic and eukaryotic cells consisting of glycoproteins glycolipids proteoglycans and glycosaminoglycans The constituents of the glycocalyx play an important role in the process of cell signaling virus transfection and immunity However detection tools for the research of glycobiology are currently in very limited supply Raybiotech has pioneered the development of antibody arrays which are now widely applied in the research community with thousands of peer reviewed publications including Cell and Nature Taking advantage of advancements in microarray technology developed for antibody arrays we have developed the largest commercially available glycan array for screening protein carbohydrate interactions This array will help researchers 1 identify the glycans binding partners in biological samples 2 identify whether target proteins are carbohydrate binding proteins 3 probe binding of viruses and whole cells to glycans 4 profile the substrate specificity of enzymes glycosyltransferases glycosidases etc 5 profile the inflammatory immune response The 100 synthetic glycans featured in the Glycan Array 100 are the most frequently identified structures showing important binding function in the literature For example influenza virus binds to a variety of sialosides with a serotype specific pattern
3. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 4 POS1 POS2 POS3 NEG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 5 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 6 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 7 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 8 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 4 42 43 44 45 46 47 48 49 50 9 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 7S 76 7 10 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 11 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 12 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 13 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 NEG POS3 POS2 POS1 14 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 NEG POS3 POS2 POS1 15 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 NEG POS3 POS2 POS1 16 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 NEG POS3 POS2 POS1 1 B Glc
4. B Sialyl Lewis A Neu5Gc a 2 3 Gal B 1 3 Fuc a 1 4 GIcNAc B Sialyl Lewis A 36 Gal a 1 4 Gal B 1 3 GIcNAc B 32 33 34 35 RayBio Glycan Array 100 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Fuc a 1 2 Gal B 1 3 GIlcNAc B 1 3 Gal B 1 4 Glc B LNFP I Fuc a 1 2 Gal B 1 4 Glc B Blood H antigen trisaccharide Gal a 1 3 Fuc a 1 2 Gal B 1 4 Glc B Blood B antigen tetrasaccharide Fuc a 1 2 Gal B 1 4 Fuc a 1 3 GIcNAc Lewis Y Fuc a 1 2 Gal B 1 3 Fuc a 1 4 GIcNAc Lewis B Gal B 1 3 Fuc a 1 4 GIcNAc B 1 3 Gal B 1 4 Fuc a 1 4 Glc B Lewis A Gal B 1 3 GalNAc B Gal B 1 3 Neu5Ac a 2 6 GalNAc B Neu5Ac a 2 6 Gal B 1 3 GalNAc B Neu5Ac a 2 6 Gal B 1 3 Neu5Ac a 2 6 GalNAc B Neu5Ac a 2 3 Gal B 1 3 Neu5Ac a 2 6 GalNAc B Neu5Ac a 2 6 Neu5Ac a 2 3 Gal B 1 3 GalNAc B GalNAc B 1 4 Neu5Ac a 2 3 Gal B 1 4 Glc B GM2 GalNAc B 1 4 Neu5Ac a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B GD2 Gal a 1 4 Gal B 1 4 GIcNAc B B D Rha Glc a 1 4 Glc B Glc a 1 6 Glc a 1 4 Glc B Maltotriose B Glc a 1 6 Glc a 1 6 Glc B Maltotetraose B GlcNAc a 1 4 GlcA B 1 4 GIcNAc a1 4 GlcA B Maltohexaose B Maltoheptaose B Acarbose B D pentamannuronic acid B L pentaguluronic acid B 17 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Gal B 1 4 GIcNAc B 1 3 Gal B 1 4
5. Galectins which are involved in apoptosis cell adhesion and T cell activation suppression function by binding beta galactosides RayBio Glycan Array 100 References 1 2 3 4 5 6 7 8 9 10 11 12 Amonsen M Smith D F Cummings R D Air G M J Virol 2007 81 8341 8345 Paulson J C de Vries R P Virus Res 2013 178 99 113 Stencel Baerenwald J E Reiss K Reiter D M Stehle T Dermody T S Nat Rev Microbiol 2014 12 739 749 Park S Shin Org Lett 2007 9 1675 1678 Pourceau G Chevolot Y Goudot A Giroux F Meyer A Moules V Lina B Cecioni S Vidal S Yu H Chen X Ferraris O Praly J P Souteyrand E Vasseur J J Morvan F Chembiochem 2011 12 2071 2080 Laurent N Voglmeir J Flitsch S L Chem Commun 2008 4400 4412 Xu R de Vries R P Zhu X Nycholat C M McBride R Yu W Paulson J C Wilson I A Science 2013 342 1230 1235 Xu R McBride R Nycholat C M Paulson J C Wilson A J Virol 2012 86 982 990 Stevens J Chen L M Carney P J Garten R Foust A Le J Pokorny B A Manojkumar R Silverman J Devis R Rhea K Xu X Bucher D J Paulson J C Cox N J Klimov A Donis R O J Virol 2010 84 8287 8299 Stowell S R Arthur C M Mehta P Slanina K A Blixt O Leffler H Smith D F Cummings R D J Biol Chem 2008 283 101
6. P y Where P1 mean signal intensity of POS spots on reference array P y mean signal intensity of POS spots on Array y X y mean signal intensity for spot X on Array y X Ny normalized signal intensity for spot X on Array y The RayBio Analysis Tool software is available for use with data obtained using RayBio Glycan Array 100 You can copy and paste your signal intensity data with and without background into the Analysis Tool and it will automatically normalize signal intensities to the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info raybiotech com for more information E Threshold of significant difference in samples After subtracting background signals and normalization to Positive Controls comparison of signal intensities between and among array images can be used to determine relative differences between samples or groups Any 21 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 RayBio Glycan Array 100 Vil Troubleshooting Guide Problem Weak Signal Cause Inadequate detection Inadequate reagent volumes or improper dilution Recommendation Increase laser power and PMT parameters Check pipet
7. of 1x Wash Buffer at room temperature with gentle shaking Completely remove wash buffer between each wash step c Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1x Wash Buffer cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min d Decant the 1x Wash Buffer from each well wash 2 times 5 min each with 800 ul of 1x Wash Buffer II at room temperature with RayBio Glycan Array 100 12 gentle shaking Completely remove wash buffer between each wash step Note Incomplete removal of the wash buffer after each wash step may cause dark spots i e background signal higher than that of the spot E Incubation with Biotinylated Detection Antibody provided by researcher Note For the Label Based protocol go directly to F Incubation with Cy3 Equivalent Dye Streptavidin step 17 on page 13 Do not do steps 13 16 13 If the researcher wishes to use their own antibody to detect specific bound proteins we recommend using a biotinylated antibody at a dilution appropriate for Western blot Optimal dilution must be determined by the researcher 14 Dilute the detection antibody in Sample Diluent Mix well and spin briefly 15 Add 400 ul of the detection antibody to each well Incubate at room temperature for 1 2 hours Note Longer incubation time is preferable for higher signals 16 Decant the samples from ea
8. ug mL RayBio Glycan Array 100 20 Blank control If scanned using optimal settings 3 distinct signal intensities will be seen POS1 gt POS2 gt POS3 If all of these signals are of similar intensity try increasing or decreasing laser power and or signal gain settings C Background Subtraction Once you have obtained fluorescence intensity data you should subtract the background and normalize to the Positive Control signals before proceeding to analysis Most laser fluorescence scanner software has an option to automatically measure the local background around each spot For best results we recommend comparing signal intensities representing the MEDIAN background signals minus local background If your resulting fluorescence signal intensity reports do not include these values e g a column labeled as MED532 B532 you may need to subtract the background manually or change the default settings on your scanner s data report menu D Normalization of Array Data To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice is arbitrary For example in our Analysis Tool Software described below the array represented by data entered in the left most column each worksheet is the default reference array RayBio Glycan Array 100 Sample Diluent only Item E 21 You can calculate the normalized values as follows X Ny X y P1
9. 09 10123 Stowell S R Arthur C M Slanina K A Horton J R Smith D F Cummings R D J Biol Chem 2008 283 20547 20559 Carlsson S Oberg C T Carlsson M C Sundin A Nilsson U J Smith D Cummings R D Almkvist J Karlsson A Leffler H Glycobiology 2007 17 663 676 RayBio Glycan Array 100 How It Works Label based Procedure Sandwich based Procedure 1A Incubation with sample rra Incubation with t en 2 3 hrs 00 8 Labeled streptavidin Incubation with thi x 800 labeled streptavidin Incubation with 1hr labeled streptavidin eS EEG 2 3hrs E Incubation with biotinylated antibody Eh Detection of signals Detection of signals Data analysis 1 IE Data analysis and graph k RayBio Glycan Array 100 ll Materials Provided Upon receipt all components of the RayBiotech Glycan Array 100 kit should be stored at 20 C After initial use remaining reagents should be stored at 4 C to avoid repeated freeze thaw cycles and may be stored for up to 3 months Labeling Reagent Item B should be prepared fresh each time before use Unused glass slides should be kept at 20 C and repeated freeze thaw cycles should be avoided slides may be stored for up to 6 months The entire kit should be used within 6 months of purchase Components Item Description 1 Slide kit 2 Slide kit Dialysis Vials Labeling Reagent Stop Solution Glycan Array Glass Slide Ass
10. Dialysis of Sample Note Samples must be dialyzed prior to biotin labeling Steps 5 7 1 To prepare dialysis buffer 1X PBS pH 8 0 dissolve 0 6 g KCI 24 g NaCl 0 6 g KH2PO and 3 45 g Na gt HPO in 2500 ml ddH20 Adjust pH 8 0 with 1M NaOH and adjust final volume to 3000 ml with ddH O 2 Add each sample into a separate Dialysis Tube Item A Load 200 ul cell culture supernatant or 100 ul cell lysates or tissue lysate 1 2 mg ml total protein or 20 ul serum or plasma 80 ul 1X PBS pH 8 5 fold dilution Carefully place Dialysis Tubes into Floating Dialysis Rack Item L Note If the samples appear to be cloudy transfer the samples to a clean tube centrifuge at 13 000 rpm for 20 minutes at 2 8 C If the samples are still not clear store them at 20 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 3 Place Floating Dialysis Rack into 2500 ml dialysis buffer in a large beaker Place beaker on a stir plate and dialyze for at least 3 hours at 4C Stirring buffer gently Then exchange the 1X PBS buffer and repeat dialysis for at least 3 hours at 4 C Transfer dialyzed sample to a clean eppendorf tube Spin dialyzed samples for 5 min at 10 000 rpm to remove any particulates or precipitates and then transfer the supernatants to a clean tube Note The sample volume may change during dialysis Note Dialysis procedure may proceed overnight RayBio Glycan Array 100
11. Glc B LNnT GlcA B 1 4 GlcNAc a 1 4 GlcA B GIcNAc B 1 6 Gal B 1 3 GalNAc a O Ser Neu5Ac a 2 3Gal B 1 4 6S GIcNAc B GalNAc B 1 4 GIcNAc B NeuS5Ac a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B Neu5SGc a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B GalNAc a 1 3 Fuc a 1 2 Gal B 1 4 Glc B Blood A antigen tetrose GlcNAc B 1 2 Man a NeuSAc a 2 3 Gal Gal B 1 3 GalNAc B 1 3 Gal B Glc a 1 2 Gal a Gal B 1 4 Fuc a 1 3 GIcNAc B 1 3 Gal B NeuSAc a 2 3 Gal 1 4 Fuc a 1 3 Glc B 3 Sialyl 3 fucosyllactose F SL RayBio Glycan Array 100 87 88 89 90 91 92 93 94 95 96 97 98 99 D cellose B Gal a 1 3 Gal B 1 4 Xylotetrose Chitin trisaccharide KDN a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B Neu5Ac a 2 8 Neu5Gc a 2 3 Gal B 1 4 Glc B Neu5Ac a 2 8 Neu5Ac a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B Neu5Ac a 2 8 Neu5Ac a 2 8 Neu5Ac a 2 8 Neu5Ac a 2 3 Gal B 1 4 Glc B Gal B 1 3 GalNAc B 1 4 Neu5Ac a 2 3 Gal B 1 4 Glc B Gentamicin Sulfate Kanamycin sulfate Geneticin Disulfate Salt G418 Neomycin trisulfate 100 SGP 18 VI Interpretation of Results and Two Examples A Explanation of Controls Spots 1 2 Positive Control spots POS1 POS2 POS3 are standardized amounts of biotinylated IgGs printed directly onto the array All other variables being equal the Positive Control intensities will be the same for each sub array This allows for normalization based upon the relative flu
12. Labeling Buffer Item K for a total volume of 300 ul Then add 3 3 ul of 1X Labeling Reagent Solution Note To normalize serum plasma or cell tissue lysate concentrations during biotinylation measure sample volume before and after dialysis Then RayBio Glycan Array 100 10 adjust the volumes of dialyzed serum plasma or cell tissue Iysates and Labeling Buffer to compensate For example if the sample volume doubles after dialysis then use twice as much serum plasma in the labeling reaction 70 ul and reduce the Labeling Buffer to 120 ul 6 Incubate the reaction solution at room temperature with gentle rocking or shaking for 30 min Mix the reaction solution by gently tapping the tube every 5 min 7 Add 3 ul Stop Solution Item C into each reaction tube and immediately dialyze as directed in Steps 1 3 on pages 9 10 Note Biotinylated samples can be stored at 20 C or 80 C until you are ready to proceed with the assay C Dry the Glass Slide Note Sandwich Based protocol starts here 8 Take out the package containing the Glycan Array Glass Slide Assembly Item D and let the slide equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry at room temperature for another 1 2 hours Do not disassemble the Glass Slide from the chamber assembly Note Protect the slide from dust or other contaminants D Blocking and Incu
13. Note Determine the total protein concentration for cell culture supernatants or cell tissue Iysate after dialysis procedure Step 3 We recommended using a BCA total protein assay eg RayBiotech Catalog 68QT BCAPro S1000 B Biotin labeling Sample Note Amines e g Tris glycine and azides quench the biotinylation reaction Avoid contaminating samples with these chemicals prior to biotinylation 4 Immediately before use prepare 1X Labeling Reagent Briefly spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the tube pipette up and down or vortex slightly to dissolve the lyophilized reagent 5 Add 1X Labeling Reagent to dialyzed samples a For labeling cell culture supernatants transfer 180 ul dialyzed sample into a new tube Add 36 ul of 1X Labeling Reagent Solution per 1 mg total protein in dialyzed cell culture supernatant Mix well For example if sample s total protein concentration is 0 5 mg ml you need to add 3 24 ul 1X Labeling Reagent to the tube of 180 ul dialyzed sample Note You need to biotin label 360 ul of dialyzed sample if dilution of the biotin labeled samples is 2 fold in step 11 on page 12 b For labeling serum or plasma Add 22 ul of 1X Labeling Reagent Solution into a new tube containing 35 ul dialyzed serum or plasma sample and 155 ul Labeling Buffer Item K c For labeling cell or tissue lysates transfer 30 ug 15 ul of 2 mg ml cell or tissue lysates into a tube and add
14. RayBio Glycan Array 100 Patent Pending Technology User Manual revised June 24 2015 Identification of the specific glycan binding proteins in serum plasma cell culture supernatants cell tissue lysates or other body fluids Cat GA Glycan 100 1 4 Sample Kit Cat GA Glycan 100 2 8 Sample Kit Cat GA Glycan 100 4 16 Sample Kit Please read this manual carefully before starting your experiment RayBiotech ea The protein array pioneer ISO 13485 Certified Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com TABLE OF CONTENTS DNYETVIEN en ee 1 Introduction nee een 2 EOW EWOTKS nen ee nee 3 Il Materials Provided 4 Ill General Considerations uaeeeee 5 A Label Based vs Sandwich Based Method 5 B Preparation of SAMPLES comune 5 C Handling Glass Slid S 6 D lINEUbat ones een 7 IV POE OC ON hanes ee ee 8 A Dialysis of Sample anne ee u 8 B Biotin labeling Sample 9 C Diy the Glass Slide sec en cee era een 10 D Blocking and Incubation 10 E Incubation with Detection Antibody Cocktail 12 F Incubation with Cy3 Equivalent Dye Streptavidin 12 G Fluorescence Detection 13 H Data ANAYSSI Saena tpt tet ttsaed lore it arte 15 Ve Glycan ArrayMapesueueuseaun anne 17 VI Interpretation of Results and Two Examples u 19 VII Troubleshooting Guide 23 RayBio Glycan Array 100 l
15. an Array 100 Glass Slide Two or four identical sub arrays on one slide 4 Subarray RayBio Glycan Array 100 E Incubations and Washes Cover the incubation chamber with adhesive film during incubation to prevent evaporation particularly when incubation is more than 2 hours Avoid foaming during incubation steps and wash steps Be sure to remove all bubbles from the sub array surface Perform all incubation and wash steps with gentle rocking motion 0 5 to 1 cycle sec 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 Several incubation steps such as step 11 sample incubation step 15 detection antibody incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation Unlike most Cy3 fluors the HiLyte Plus 555 used in this kit is very stable at room temperature RT and resistant to photobleaching on the hybridized glass slides However please protect glass slides from directly strong light and temperatures above RT RayBio Glycan Array 100 IV Protocol READ ENTIRE PROTOCOL BEFORE STARTING Note Biotin Label Based protocol starts here For the Sandwich Based protocol using researcher s own detection antibody start at section C Dry the Glass Slide step 8 on page 11 Do not do steps 1 7 A
16. bation Note Glass slide should be completely dry before adding Sample Diluent to wells 9 Block sub arrays by adding 400ul Sample Diluent Item E into each well and incubate at room temperature for 30 min Ensure there are no bubbles on the array surface RayBio Glycan Array 100 10 Immediately prior to sample incubation spin biotin labeled samples for 5 min at 10 000 rpm to remove any particulates or precipitates Dilute samples with Sample Diluent Item E Note Recommended dilution of the biotin labeled samples with Sample Diluent prior to incubation is 2 10 fold for cell culture supernatants 20 fold for serum plasma or 30 fold cell tissue lysate 11 Decant buffer from each well Add 400ul of sample to each well Incubate arrays with gentle rocking or shaking at room temperature for 2 3 hours Longer incubation time is preferable if higher signal intensity is desired Note This step may be done overnight at 4 C for highest intensities 12 Wash a Based on number of samples and remaining protocol calculate the amounts of 1x Wash Buffers amp Il that are needed for each step of the protocol Separately dilute required amounts of 20x Wash Buffer I and 20x Wash Buffer II with ddH O to 1x concentration For example if 12 ml of 1x Wash Buffer is needed then 600 ul of 20x Wash Buffer would be diluted to a final volume of 12 ml b Decant the samples from each well and wash each well 5 times 5 min each with 800 ul
17. ch well and wash 5 times with 800 ul of 1x Wash Buffer and then 2 times with 800 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer between each wash step F Incubation with Cy3 Equivalent Dye Streptavidin 17 Prepare 1X Dye conjugated Streptavidin a Briefly spin down the Cy3 equivalent dye conjugated streptavidin tube Item H immediately before use b Add 1000 ul of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently do not store the stock solution for later use RayBio Glycan Array 100 13 c Add 400 ul of Cy3 Conjugated Streptavidin stock solution into a tube with 1400 ul of Blocking Buffer Mix gently to prepare 1X Cy3 Conjugated Streptavidin 18 Add 400 ul of 1X Cy3 equivalent dye conjugated streptavidin to each well Cover the incubation chamber with the plastic adhesive strips Item J and cover the slide with aluminum foil to avoid exposure to light or incubate in dark room 19 Incubate the slide with Cy3 Conjugated Streptavidin at RT for 1 hour with gentle rocking or shaking 20 Decant the samples from each well and wash 5 times with 800 ul of 1x Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step G Fluorescence Detection 21 Disassemble the slide assembly by pushing clips outward from the slide side as shown below Carefully remove the slide from the gasket Note Be careful not to t
18. embly 1 Slide 2 Slides Sample Diluent 1 1 20X Wash Buffer 20X Wash Buffer II Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Labeling Buffer Floating Dialysis Rack Manual Each slide contains 4 identical subarrays HiLyte Plus 555 Additional Materials Required e Detection antibodies of interest For sandwich based method only e Distilled or de ionized water Orbital shaker e Laser scanner for fluorescence detection RayBio Glycan Array 100 Aluminum foil Small plastic or glass containers 1 5 mL Polypropylene microcentrifuge tubes KCI NaCl KH2PO and Na gt HPO For label based method only Beaker stir plate and stir bar Pipettors pipette tips and other common lab consumables General Considerations A Label Based vs Sandwich Based Method The RayBiotech Glycan Array 100 Kit can be used with either a label based method or with a sandwich based method e Inthe label based method the proteins or antibodies in the sample are biotin labeled via a simple reaction targeting primary amines allowing direct detection on the array via a cy3 equivalent dye conjugated biotin streptavidin complex A complete protocol and the primary materials for this procedure are included with the kit e The sandwich based method is used for antibody based detection of target proteins captured on the array The user will need to supply the labeled reporter antibodies specific for the
19. er by following step as shown in the figures below To avoid breaking the printed glass slide you may first want to practice assembling the device with a blank glass slide 1 Apply slide to incubation chamber barcode facing upward as in image A below 2 Gently snap one edge of a snap on side as shown in image B 3 Gently press other of side against lab bench and push in lengthwise direction image C 4 Repeat with the other side image D A RayBio Glycan Array 100 15 H Data Analysis Data extraction can be done with most of the microarray analysis software GenePix ScanArray Express ArrayVision or MicroVigene NOTE Due to the difficulty of printing glycans each glycan is printed in quadruplicate We guarantee each glycan to have at least 3 spots present and additionally guarantee no more than 5 total spots per array will be missing V Glycan Array Map Glycan structures and molecular weights are available at this link www raybiotech com GlycanArray100Structures 100 Glycan Array Map 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 POS1 POS2 POS3 NEG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2 POS1 POS2 POS3 NEG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 3 POS1 POS2 POS3 NEG 1 2
20. ir protein of interest An example protocol for this procedure with a general Antibody Cocktail is included in this manual Specific antibody concentrations and conditions will need to be determined by the end user B Preparation of Samples e We recommend the following parameters for your samples o 300 to 400 ul of 40X diluted serum plasma cell culture media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Note If you experience high background or the readings exceed the detection range further dilution of your sample is recommended RayBio Glycan Array 100 C Handling Glass Slides e The microarray slides are delicate Please do not touch the surface of the slides with pipette tips forceps or your fingers Hold the slides by the edges only e Handle the slides with latex free gloves in a clean environment e Do not remove the glass slide from the chamber assembly until step 21 and take great care not to break the glass slide when doing so e Because there is no barcode on the slide transcribe the slide serial number from the slide bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide s frame is removed this will allow you to distinguish one slide from another e Remove reagents sample by gently applying suction with a pipette to corners of each chamber Do not touch the printed area of the array only the sides D Layout of Glyc
21. ly and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price 2015 RayBiotech Inc RayBio Glycan Array 100 24
22. orescence signal responses to a known control much as housekeeping genes or proteins are used to normalize results in PCR or Western blots respectively Negative Control NEG spots contain a protein containing buffer used to dilute glycans printed on the array Their signal intensities represent non specific binding of Biotin conjugated anti Cytokines and or the Cy3 Conjugated Streptavidin Negative control signal intensities are usually very close to background signals in each sub array RayBio Glycan Array 100 19 B Typical Results Obtained with RayBio Glycan Array 100 The following figure shows the RayBio Glycan Array 100 probed with biotin labeled lectin mixtures The images were captured using a Axon GenePix laser scanner The strong signals in the upper left and lower right corners of each array are Positive Controls which can be used to identify the orientation and help normalize the results between arrays In sample 1 glycans 6 7 27 51 54 and 90 showed strong binding activities to biotin lectins b BSL I b WGA indicating that those glycans contain aGal aGalNAc and GIcNAc groups in their structures In sample 2 glycans 3 50 58 and 61 showed stronger signals than in sample 1 meaning that these glycans have aFuc groups in their structures Sample 1 Biotin lectin mixture b BSL I b WGA 0 4 ug mL Sample 2 Biotin lectin mixture b BSL I b WGA b UEA 0 4 g mL and b Con A 0 04
23. ouch the surface of the array 22 Gently place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube Item I add enough 1x Wash Buffer about 30 ml to cover the whole slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer I Wash with 1x Wash Buffer II about 30 ml and gently shake at room temperature for 5 minutes 23 Finally wash the glass slide with 30 mL of de ionized or distilled water for 5 min RayBio Glycan Array 100 14 24 Remove water droplets completely by gently applying suction with a pipette Do not touch the sub array areas only the sides of the slide Make sure the finished glass slide is completely dry before scanning or storage 25 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength such as Axon GenePix Make sure that the signal from the spot containing the highest concentration receives the highest possible reading yet remains unsaturated Note Unlike most Cy3 fluors the HiLyte Plus Fluor 555 used in this kit is very stable at RT and resistant to photobleaching on completed glass slides However please protect glass slides from temperatures above RT and store them in the dark Do not expose glass slide to strong light such as sunlight or UV lamp Note If you need to repeat any of the incubation after finishing the experiment you must first re assemble the glass slide into the incubation chamb
24. tes and ensure correct preparation Short incubation time Ensure sufficient incubation time or change sample incubation step to overnight Too low glycan concentration in sample Reduce amount of dilution or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Uneven Signal Bubble formed during incubation Arrays are not completed covered by reagent Handle and pipette solutions more gently De gas solutions prior to use Prepare more reagent and completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer Comet tail formation Air dry the slide for at least 1 hour before usage Generel Inadequate detection Increase laser power that the highest concentration for each lectin receives the highest possible reading yet remains unsaturated Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High Insufficient wash Increase wash time and use more wash buffer Background Dust Minimize dust in work environment before starting experiment Slide is allowed to dry out Take additional precautions to prevent slides from dying out during experiment RayBio Glycan Array 100 23 Note This product is intended for research on
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