RayBiotech, Inc. RayBiotech Lectin Array 40
Contents
1. if higher signal intensity is desired Note We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation if less than 70 ul of sample or reagent is used Note This step may be done overnight at 4 C for highest intensities 11 Wash RayBiotech Lectin Array 40 Kit 10 a Calculate the amounts of 1x Wash Buffers amp I that are needed for each step of the protocol Separately dilute required amounts of 20x Wash Buffer and 20x Wash Buffer II with ddH5 O to 1x concentration For example if 12 ml of 1x Wash Buffer I 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 b min each with 150 ul 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 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 150 ul of 1x Wash Buffer Il at room temperature with gentle shaking Completely remove wash buffer between each wash step Note Incomplete removal of the wash bu2. 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 Lectin Array 40 Map Pos ASA ConA EEL GSI LBA NPA SBA SNAJ UEA RayBiotech Lectin Array 40 Kit 13 V Lectin Array 40 Key ledin Abbreviation Source Carbohydrate Specificity _ Griffonia Bandeiraea simplicifolia GS I GSL I BSL I Griffonia Bandeiraea simplicifolia seeds aGal aGalNAc GS 11 SLA BSL Hippeastrum hybrid Hippeastrum hybrid Amaryllis bulbs edi Abbreviation Source Carbohydrate Specificity _ Winged Pea Asparagus Pea seeds j NAcD2M IcNAcb4 FRESEOMIS vigarie PHA E Phaseolus vulgaris Red Kidney Bean seeds Sapaal cach Man Toy GENAD Erythroagglutinin GICNAcB4Mana3 ManB4 Leucoagglutinin PHA L Phaseolus vulgaris Red Kidney Bean seeds Gal B4GI cNAcB6 GICNAcB2Mana3 Mana3 Psophocarpus PTL PTL I WBA I Psophocarpus tetragonolobus Winged Bean seeds Gal NAc Gal NANA 2 INAc gt GalNAc Lac gt Sambucus nigra SNA I Sambucus nigra Elderberry bark a 2 6 Gal NAc gt GalNAc Lac Gal NANAa 2 6 Gal Sugar Abbreviations Fuc L Fucose Gal D Galactose GalNAc N Acetylglactosamine Glc D Glucose GIcNAc N Acetylglucosamine Lac Lactose Man Mannose RayBiotech Lectin Array 40 Kit 14 VI Application 1 Detection of Glycans o
3. RayBiotech Lectin Array 40 Detect glycan profiles using 40 lectins User Manual Version 1 5 Updated May 10 2015 Cat GA Lectin 40 RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www RayBiotech com Email info RayBiotech com AAA AIA ASA BPA DSA ECA GNA GS I GS Il HHA LBA LcH A LEA Lotus MAA NPA PHA E PHA L PSA SJA SNA I SNA II UEA IIl VFA WFA ACL Con A SBA WGA DBA UEA I PNA Jacalin STL VVA EEL MPL PILI AAL UDA One standard glass slide is spotted with 8 wells of Format identical lectin sub arrays Each lectin is printed in duplicated on every sub array Lectins printed on slides 40 Detection Method Fluorescence with laser scanner Cy3 equivalent dye Sample Volume 50 100 ul per array Reproducibility CV 2096 EY PA See Section For Array co D 4 ez a NS a Ex e NS a NS a NS e A Fluor dye cy3 Biotin Streptavidin Glycan from Lecti Glass Slide RayBiotech Lectin Array 40 Kit 1 TABLE OF CONTENTS VI VII VIII OVEIVIC W oa EEE A NETO OEE E et WEE OCULI OTL sensuna R How It Works Materials Provided t General Considerations e A Label Based vs Sandwich Based Method B Preparation of Samples C Handling Glass Slides E JDnoubdblo scende opm M LO P AE UEM PREIE CLE RR Onmmoa
4. at 1 000 rom for 3 minutes without cap ii Or dry the glass slide by a compressed N stream ii Or gently apply suction with a pipette to remove water droplets Do not touch the sub array areas only the sides of the slide 19 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 If the signal intensity for different lectins vary greatly in the same array we recommend using multiple scans with a higher PMT for low signal lectins and a low PMT for high signal lectins RayBiotech Lectin Array 40 Kit 12 G Data Analysis 20 Data extraction can be done using the GAL file that is specific for this array along with the microarray software commonly available in most microarray laser scanners GenePix ScanArray Express etc GAL files can be found on our website here www RayBiotech com Gal Files html H Normalization of Array Data 21 To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice can be arbitrary For example in our Analysis Tool Software the array represented by data entered in the left most column each worksheet is the default reference array You can calculate the normalized values as follows X Ny X y P1 P y Where
5. ein concentration for cell culture supernatants or cell tissue lysate after dialysis procedure Step 3 We recommended using a BCA total protein assay eg Pierce Catalog 23227 RayBiotech Lectin Array 40 Kit 8 B Biotin Labeling of 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 2 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 3 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 labeling bu
6. erest has glycan moieties and 3 find specific glycan binding ligands in biological samples Lectins are glycan binding proteins which have been purified from trees beans and some fruits They are highly specific for a given glycan based on their sequence and the different sugar unit structures the glycan contains For the RayBiotech lectin array one standard glass slide is spotted with 8 wells of identical lectin arrays Each lectin together with the positive controls is arrayed in duplicate The slide comes with a 8 well removable gasket which allows for the process of 8 samples using one slide Four slide slides can be nested into a tray which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously The RayBiotech lectin array provides a powerful new tool for glycosylation determination drug discovery and biomarker development all with limited samples volumes required RayBiotech Lectin Array 40 Kit 3 How It Works Label based Approach T rrA laam Biotin sample E Labeled streptavidin c E o with Labeled 1hr streptavidin a Detection of signals lt j pon E Data analysis and graph Le RayBiotech Lectin Array 40 Kit Sandwich based Approach IIA zares Sample incubation E AA with 1 2 hr m Ab p Incubation with Labeled 9 oe ihr streptavidin Detection of signals a C EE NN S Data analysi
7. ffer Item 3 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 adjust the volumes of dialyzed serum plasma or cell tissue lysates and Labeling Buffer to compensate RayBiotech Lectin Array 40 Kit 9 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 4 into each reaction tube and immediately dialyze as directed in Steps 1 3 on page 8 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 8 Take out the glass slide containing bag from the box 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 Note Incomplete drying of slides before use may cause the formation of comet tails D Blocking and Incubation 9 Add 100ul Sample Diluent Item 6 into each well and incubate at room temperature for 30 min to block slides 10 Decant buffer from each well Add 100ul of sample to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable
8. ffer after each wash step may cause dark spots Background signal is higher than that of the spot E Incubation with Cy3 Equivalent Dye Streptavidin 12 Briefly spin down the Cy3 equivalent dye conjugated streptavidin tube 13 Add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 14 Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the slide with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for 1 hour RayBiotech Lectin Array 40 Kit 11 15 Decant the samples from each well and wash 5 times with 150 ul of 1x Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step F Fluorescence Detection 16 Disassemble the slide assembly by pushing clips outward from the Slide side Carefully remove the slide from the gasket Note Be careful not to touch the surface of the array 17 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube 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 with gentle and gently shake at room temperature for 5 minutes 18 Remove water droplets completely by one of the following ways i Put the glass slide into the Slide Washer Dryer and dry the glass slide by centrifuge
9. ide for at least 1 hour before usage 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 Insufficient wash Increase wash time and use more wash High buffer Background NONU l Dust Minimize dust in work environment before A a nnd slides from dying out during experiment RayBiotech Lectin Array 40 Kit 18 Note This product is intended for research only 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 This product is for research use only 2010 RayBiotech Inc RayBiotech Lectin Array 40 Kit 19
10. le is recommended RayBiotech Lectin Array 40 Kit 6 C Handling Glass Slides Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass slide in clean environment 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 frame assembly is removed this will allow you to distinguish one slide from another D Incubation A B C D Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation to prevent evaporation particularly when incubation is more than 2 hours or lt 70 ul of sample or reagent is used Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation RayBiotech Lectin Array 40 Kit 7 IV Protocol READ ENTIRE PROTOCOL BEFORE STARTING A Dialysis of Sample Note For the Sandwich based protocol start at C Drying the Glass Slide step 8
11. n a Purified Protein In this application the purified protein Horseradish Peroxidase HRP was investigated After biotinylation of HRP we can determine what kind of glycans are conjugated to HRP by using the RayBiotech lectin array Lectins GNA HHA NPA showed strong signals after incubation with 0 33 ug mL Biotin HRP followed detection by streptavidin fluorescence dye Figure A B and C The fluorescence signals from GNA HHA and NPA can be blocked in a concentration depend manner by HRP itself Figure A and C which means that these fluorescence signals were generated based on the binding between HRP and the three lectins As we know GNA HHA and NPA lectins specifically bind to mannose which indicates that HRP contains mannose By adding increasing amount of mannose the signal from GNA HHA and NPA can be reduced Figure A and B The reduction signals from increasing concentrations of mannose confirms that HRP protein contains mannose in its glycocalyx Lectin VVA binds to the streptavidin fluorescence dye A Biotin HRP 0 33 ug ml Mannose O ug mL GNA gt HHA C5 NPA CO RayBiotech Lectin Array 40 Kit 15 B Mannose C HRP 30000 30000 25000 300 mM 25000 Mannose 20000 20000 30 mM Mannose E OOUR MENRE 15000 15000 E 10 ug mL HRP 10000 3mM Mannose 10000 E 1 ug mL HRP 0 TT EN E EZ 0 4 T T T E 1 Ac A amp N
12. o WwW D H Normalization of Array Data s Lectin Array 40 Map Lectin Array 40 Key Application 1 Detection of Glycans on HRP Application 2 Profile of Serum Sample Other Applications Troubleshooting Guide RayBiotech Lectin Array 40 Kit Dialysis of Sample Biotin labeling Sample Dry the Glass Slide Blocking and Incubation Incubation with Cy3 Equivalent Dye Streptavidin Fluorescence Detection iis PDA TANU S RE O O N N N DD DW WN A W e e e e e e e e RP RP h HB CON OD aa A W U U N e O O I 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 pioneered the development of antibody arrays which are now widely applied in the research community with hundreds of peer reviewed publications such as in Cell and Nature Taking advantage of advancements in microarray technology developed for antibody arrays we recently developed glycan arrays to help researchers 1 identify and profile the glycans in their samples 2 determine whether their biomarker of int
13. on page 10 Do not do steps 1 7 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 KH PO and 3 45 g Na HPO in 2500 ml ddH O 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 Vial Item 1 Load 200 ul cell culture supernatant or 100 ul cell lysates or tissue lysate 172 mg ml total protein or 20 ul serum or plasma 80 ul 1X PBS pH 8 5 fold dilution Carefully place Dialysis Vials into Floating Dialysis Rack 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 precipitants and then transfer the supernatants to a clean tube Note The sample volume may change during dialysis Note Dialysis procedure may proceed overnight Note Determine the total prot
14. ov of O 5000 Qo 2 e CS S 5999 POS Neg GNAHHANPA Vil Application 2 Profiling of a Serum Sample Using the lectin array we can discover the different glycoprotein profiles of the serum samples or cell lysates from patient cohorts versus a control group Below images showed the profiles of the glycans from serum samples detected by Biotin anti human IgG and Fluorescence dye streptavidin E EI Different serum samples RayBiotech Lectin Array 40 Kit 16 Different serum samples No serum sample VIII Other Applications Quantitative analysis of lectin glycoprotein interactions Example a concentration series of glycoproteins detected with the lectin array could reveal concentration dependent effects of lectin glycan binding Determine the profile of bacterial cell surface glycans Cell lysate from bacteria can be Biotinylated and hybridized to the lectin array Analysis of the binding pattern and correlation with the known carbohydrate binding specificities of the lectins can determine the glycans on the cell membrane RayBiotech Lectin Array 40 Kit 17 IX Troubleshooting Guide Problem Cause Recommendation improper dilution preparation Weak Signal sample incubation step to overnight sample sample freeze thaw the slide De gas solutions prior to use reagent cover arrays with solution adhesive film during incubation Cross contamination from Avoid overflowing wash buffer neighboring wells Air dry the sl
15. s A Label Based vs Sandwich Based Method The RayBiotech Lectin Array 40 Kit can be used with either a label based method or as a sandwich based method e The label based method is used to biotin samples containing proteoglycans and glycoproteins for 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 The sandwich based method is used for antibody detection of glycocalyx elements glycolipids glycoproteins etc captured on the array The user will need to supply the labeled reporter antibodies specific for the glycocalyx elements 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 Use serum free conditioned media if possible If serum containing conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contain glycocalyx elements We recommend the following parameters for your samples o 50 to 100 ul of original or 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 samp
16. s and graph LJ IE ll Materials Provided Upon receipt all components of the RayBiotech Lectin Array 40 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 2 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 4 Slide kit Dialysis Vials Labeling Reagent Labeling Buffer Stop Solution Lectin Array Glass Slide Assembly Sample Diluent 20X Wash Buffer 20X Wash Buffer II Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Floating Dialysis Rack Manual I Im o WW N erererrererere A NPP rPNN DN OO 2 3 4 5 6 7 8 9 PARR PR Additional Materials Required e Detection antibodies of interest For sandwich based method only e Orbital shaker e aser scanner for fluorescence detection e Aluminum foil e 1 5ml Polypropylene microcentrifuge tubes e KCl NaCl KH PO and Na HPO For label based method only e Plastic or glass containers beaker stir plate and stir bar e Pipettors pipette tips ddH O and other common lab consumables RayBiotech Lectin Array 40 Kit 5 General Consideration
Download Pdf Manuals
Related Search