Manual - RayBiotech, Inc.
Contents
1. HyLite Plus is a trademark of Anaspec Inc D e D D D GenePix is a registered trademark of Molecular Devices Inc RayBio L Series Mouse Antibody Array L 308 Protocol 27 This product is for research use oniv 2011 RayBiotech Inc RayBio L Series Mouse Antibody Array L 308 Protocol 282. 246 247 247 248 248 249 249 250 250 251 251 252 252 19 253 253 254 254 255 255 256 256 257 257 258 258 259 259 260 260 261 261 262 262 263 263 264 264 265 265 266 266 20 267 267 268 268 269 269 270 270 271 271 272 272 273 273 274 274 275 275 276 276 277 277 278 278 279 279 280 280 21 281 281 282 282 283 283 284 284 285 285 286 286 287 287 288 288 289 289 290 290 291 291 292 292 293 293 294 294 22 295 295 296 296 297 297 298 298 299 299 300 300 301 301 302 302 303 303 304 304 305 305 306 306 307 307 308 308 23 309 309 310 310 311 311 3127 312 313 313 314 s14 as 315 316 316 Neg Neg Neg Neg Neg Neg P 3c P 30 P zc P 2c Pc P te RavBio L Series Mouse Antibodv Arrav L 308 Protocol 18 RayBio L series Mouse Antibody Array L 308 List Number Name Number Name Number Name Number Name Number Name Number Name 1 Positive 1a 57 CXCL16 113 Granzyme D 169 IL 12 R beta 1 225 MIP 2 281 TIMP 2 2 Positive 2a 58 CXCR2 IL 8 RB 114 Granzyme G 170 IL 13 226 MIP 3 alpha 282 TIMP 4 3 Positive 3a 59
3. CXCR3 115 Gremlin 171 IL 13 R alpha 2 227 MIP 3 beta 283 TLIA TNFSF15 4 neg 60 CXCR4 116 Growth Hormone R 172 IL 15 228 MMP 2 284 TLR1 5 6Ckine 61 CXCR6 117 HGF R 173 IL 15 R alpha 229 MMP 3 285 TLR2 6 Activin A 62 DAN 118 HGF 174 IL 16 230 MMP 9 286 TLR3 7 Activin C 63 Decorin 119 HVEM TNFRSF14 175 IL 17 231 MMP 12 287 TLR4 8 Activin RIB ALK 4 64 DKK 1 120 ICAM 1 176 IL 17BR 232 MMP 14 LEM 2 288 TMEFF1 Tomoregulin 1 9 Adiponectin Acrp30 65 Dkk 3 121 ICAM 2 CD102 177 IL 17C 233 MMP 24 MTS MMP 289 TNF RI TNFRSF1A 10 AgRP 66 Dkk 4 122 ICAM 5 178 IL 17D 234 Neuregulin 3 NRG3 290 TNF Ril 11 ALCAM 67 DPPIV CD26 123 ICK 179 IL 17E 235 Neurturin 291 TNF alpha 12 Angiopoietin like 2 68 DR3 TNFRSF25 124 IFN alpha beta R1 180 IL 17F 236 NGF R TNFRSF16 292 TNF beta TNFSF1B 13 Angiopoietin like 3 69 Dtk 125 IFN alpha beta R2 181 IL 17R 237 NOV CCN3 293 TPO 14 AR Amphiregulin 70 EDAR 126 IFN beta 182 IL 17RC 238 Osteoactivin GPNMB 294 TRAIL TNFSF10 15 Artemin 71 EGFR 127 IFN gamma 183 IL 17RD 239 Osteopontin 295 TRAIL R2 TNFRSF10B 16 Axl 72 EG VEGF PK1 128 IFN gamma R1 184 IL 18 R alpha IL 1 R5 240 Osteoporotegerin 296 TRANCE TNFSF11 17 b FGF 73 Endocan 129 IGFBP 1 185 IL 20 241 0X40 Ligand TNFSF4 297 TREM 1 18 B7 1 CD80 74 Endoglin CD105 130 IGFBP 2 186 IL 20 R alpha 242 PDGFC 298 TROV 19 BAFF R TNFRSF13C 75 Endostatin 131 IGFBP 3 187 IL 21 243 PDGF R alpha 299 TSLP 20 BCMA TNFRSF17 76 Eotaxin 132 IGFBP 5 188 IL 21 R 2
4. for 5 min per wash 14 Obtain a clean container e g pipette tip box or slide staining jar place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 10 min per wash 15 Dilute 20X Wash Buffer II Concentrate Item H 20 fold with de ionized or distilled water Decant the Wash Buffer from each well place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer II to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 5 min per wash 16 Prepare 1X Cy3 Conjugated Streptavidin a Briefly spin down tube containing the Cy3 Conjugated Streptavidin Item immediately before use b Add 1000 ul of Blocking Buffer into the tube to prepare a concentrated Cy3 Conjugated Streptavidin stock solution Pipette up and down to mix gently do not store the stock solution for later use c Add 200 ul of Cy3 Conjugated Streptavidin stock solution into a tube with 800 ul of Blocking Buffer Mix gently to prepare 1X Cy3 Conjugated Streptavidin RavBio L Series Mouse Antibody Array L 308 Protocol 14 17 Carefullv remove Assembled Glass Slide from container Remove all of Wash Buffer II from the wells Add 400 ul of 1X Cy3 Conjugated Streptavidin to each sub array Cover the incubation cham
5. sample volume increased from 100 ul to 200 ul add 70 ul dialyzed serum and 120 ul Labeling Buffer to keep same total volume 212 ul 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 RavBio L Series Mouse Antibodv Arrav L 308 Protocol buffer Item K for a total volume of 300 ul Then add 3 3 ul of 1X Labeling Reagent Solution 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 D into each reaction tube and immediately dialyze as directed in Steps 2 3 Note Biotinylated samples can be stored at 20 C or 80 C until you are ready to proceed with the assay C Drying of the Glass Slide 8 Remove the package containing the Assembled Glass Slide Item E from the freezer Place unopened package on the bench top for approx 15 min and allow the Assembled Glass Slide to equilibrate to room temperature RT 9 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 RT Note Protect the slide from dust or others contaminants D Blocking and Incubations Note Glass slide should be completely dry before adding Blocking Buffer to wells RayB
6. 124 125 125 126 126 10 127 127 128 128 129 129 130 130 131 131 132 132 133 133 134 134 135 135 136 136 137 137 138 138 139 139 140 140 11 141 141 142 142 143 143 144 144 145 145 146 146 147 147 148 148 149 149 150 150 151 151 152 152 153 153 154 154 12 P tb Pb P 2b P 2b P 3b P 3b Neg Neg 159 159 160 160 161 161 162 162 163 163 164 164 165 165 166 166 167 167 168 168 13 169 169 170 170 171 171 172 172 173 173 174 174 175 175 176 176 177 177 178 178 179 179 180 180 181 181 182 182 14 183 183 184 184 185 185 186 186 187 187 188 188 189 189 190 190 191 191 192 192 193 193 194 194 195 195 196 196 15 197 197 198 198 199 199 200 200 201 201 202 202 203 203 204 204 205 205 206 206 207 207 208 208 209 209 210 210 16 211 211 212 212 213 213 214 24 215 215 216 216 217 217 218 218 219 219 220 220 221 221 222 222 223 223 224 224 17 225 225 226 226 227 227 228 228 229 229 230 230 231 231 232 232 233 233 234 234 235 235 236 236 237 237 238 238 18 239 239 240 240 241 241 242 242 243 243 244 244 245 245 246
7. 44 PDGF R beta 300 TSLPR 21 beta Catenin 77 Eotaxin 2 133 IGFBP 6 189 IL 22 245 Pentraxin3 TSG 14 301 TWEAK TNFSF12 22 BLC 78 Epigen 134 IGFBP rp1 IGFBP 7 190 IL 22BP 246 PF 4 302 TWEAK R TNFRSF12 23 BTC Betacellulin 79 Epiregulin 135 IGF I 191 IL 23 247 PIGF 2 303 Ubiqultin 24 Cardiotrophin 1 80 Ervthropoietin EPO 136 IGF II 192 IL 23 R 248 Progranulin 304 uPAR 25 CCLI 1 309 TCA 3 81 E Selectin 137 IL 1 alpha 193 IL 24 249 Prolactin 305 Urokinase 26 CCL28 82 FADD 138 IL 1 beta 194 IL 27 250 P Selectin 306 VCAM 1 27 CCL4 MIP 1 beta 83 FAM3B 139 IL 1 R4 ST2 195 IL 28 IFN lambda 251 RAGE 307 VE Cadherin 28 CCL7 MCP 3 MARC 84 Fas TNFRSF6 140 IL 1 R6 IL 1 R rp2 196 IL 31 252 RANTES 308 VEGF 29 CCL8 MCP 2 85 Fas Ligand 141 IL 1 R9 197 IL 31 RA 253 RELM beta 309 VEGF R1 30 CCR10 86 FCrRIIB CD32b 142 IL 1 RI 198 Insulin 254 Resistin 310 VEGF R2 31 CCR3 87 FGF R3 143 IL 1 RII 199 Integrin beta 2 CD18 255 S100A10 311 VEGF R3 32 CCR4 88 FGF R4 144 IL 2 200 1 TAC 256 SCF 312 VEGF B 33 CCR6 89 FGF R5 beta 145 IL 2 R alpha 201 KC 257 SCF R c kit 313 VEGFC 34 CCR7 90 FGF 21 146 IL 2 R beta 202 Kremen 1 258 SDF 1 314 VEGF D 35 CCR9 91 Fit 3 Ligand 147 IL 3 203 Kremen 2 259 Serum Amvloid A1 315 WIF 1 36 CD11b 92 FLRG Follistatin 148 IL 3 R alpha 204 Lefty 1 260 Shh N 316 WISP 1 CCN4 37 CD14 93 Follistatin like 1 149 IL 3 R beta 205 Leptin R 261 SIGIRR 317 Neg 38 CRP 94 Fractalkine 150 IL 4 206 LEPTIN OB 262 SLPI 318 Neg 39 CD27 TNFR
8. 926 940 Lin Y Huang R Chen L et al Profiling of cytokine expression by biotin labeled based protein arrays Proteomics 2003 3 1750 1757 Huang R Jiang W Yang J et al A Biotin Label based Antibody Array for High content Profiling of Protein Expression Cancer Genomics Proteomics 2010 7 3 129 141 Liu T Xue R Dong L et al Rapid determination of serological cytokine biomarkers for hepatitis B virus related hepatocellulare carcinoma using antibody arrays Acta Biochim Biophys Sin 2011 43 1 45 51 Cui J Chen Y Chou W C et al An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer Nucl Acids Res 2011 39 4 1197 1207 Jun Zhong et all Temporal Profiling of the Secretome during Adipogenesis in Humans Journal of Proteome Research 2010 9 5228 5238 Chowdury UR Madden BJ Charlesworth MC Fautsch MP Proteomic Analysis of Human Aqueous Humor Invest Ophthalmol Visual Sci 2010 51 10 4921 4931 RavBio L Series Mouse Antibody Array L 308 Protocol 25 8 Wei Y Cui C Lainscak M et al Type specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end stag heart failure patients relationshp between MMP 10 and LV remodeling J Cell Mol Med 2011 15 4 773 782 9 Kuranda K Berthon C Lep tre F et al Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem progenito
9. RayBio Label Based L Series Mouse Antibody Array 308 L 308 Patent Pending Technology User Manual Revised Jan 18 2014 For the simultaneous detection of the relative expression of 308 L 308 mouse proteins in serum plasma cell culture supernatants cell tissue lysates or other body fluids L Series Mouse Antibody Array L 308 Cat AAM BLG 1 2 2 Sample Kit Cat AAM BLG 1 4 4 Sample Kit Please read manual carefuly before starting experiment wm RayBiotech Inc Ga the protein array pioneer company Your Provider for Excellent Protein Array Systems and Services 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 RavBiotech Inc TABLE OF CONTENTS I Introduction and How It Work II Materials Provided sed aseene ee A Storage Recommendations 00an00n B Additional Materials Required II Overview and General Considerations A Preparation and Storage of Samples B Handling the Glass Slides cece ee C Glass Slide Lavout cece cece e ees D Incubation and Washes eee Ae PEO COCO Li A Dialysis of Sample eee B Biotin Labeling of Sample 4 C Drying of the Glass Chip D Blocking and Incubations eee E Fluorescence Detection cece eeeeees V Antibody Array Map VI Interpretation of Results VII Troubleshooting Guide e
10. SF7 95 Frizzled 1 151 IL 4R 207 LIF 263 Soggy 1 319 Neg 40 CD27 Ligand TNFSF7 96 Frizzled 6 152 IL 5 208 LIGHT TNFSF14 264 SPARC 320 Positive 3c 41 CD30 97 Frizzled 7 153 IL 5 R alpha 209 LIX 265 Spinesin Ectodomain 321 Positive 2c 42 CD30L 98 Galectin 3 154 IL 6 210 LRP 6 266 TACI TNFRSF13B 322 Positive 1c 43 CD40 99 G CSF 155 Positive 1b 211 L Selectin 267 TARG 323 44 CD40 Ligand TNFSF5 100 GDF 1 156 Positive 2b 212 Lungkine 268 TCA 3 324 45 Cerberus 1 101 GDF 3 157 Positive 3b 213 Lymphotactin 269 TCCR WSX 1 325 46 Chordin Like 2 102 GDF 5 158 neg 214 Lymphotoxin beta R TNFRSF3 270 TECK 326 47 Coagulation Factor III Tissue Factor 103 GDF 8 159 IL 6R 215 MAdCAM 1 271 TFPI 327 48 Common gamma Chain IL 2 R gamma 104 GDF 9 160 IL 7 216 MCP 1 272 TGF beta 1 328 49 CRG 2 105 GFR alpha 2 GDNF R alpha 2 161 IL 7 R alpha 217 MCP 5 273 TGF beta 2 329 50 Cripto 106 GFR alpha 3 GDNF R alpha 3 162 IL 9 218 M CSF 274 TGF beta 3 330 Si Crossveinless 2 107 GFR alpha 4 GDNF R alpha 4 163 IL 9R 219 MDC 275 TGF beta RI ALK 5 331 52 Cryptic 108 GITR 164 IL 10 220 MFG E8 276 TGF beta RII 332 53 Csk 109 GITR Ligand TNFSF18 165 IL 10 R alpha 221 MFRP 277 Thrombospondin 333 54 CTACK 110 Glut2 166 IL 11 222 MIG 278 Thvmus Chemokine 1 334 55 CTLA 4 CD152 111 GM CSF 167 IL 12 p40 p70 223 MIP 1 alpha 279 Tie 2 335 56 CXCL14 BRAK 112 Granzyme B 168 IL 12 p70 224 MIP 1 gamma 280 TIMP 1 336 RayBio L Series Mouse Ant
11. Sample 2 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 intensitv trv increasing or decreasing laser power and or signal gain settings Also in the absence of an external standard curve for each protein detected there is no means of assessing absolute or relative concentrations of different proteins in the same sample using immunoassavs If vou wish to obtain quantitative data ie concentrations of the various analvtes in vour samples trv using our Quantibody Arrays instead RavBio L Series Mouse Antibody Array L 308 Protocol 21 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 have 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
12. ap A RayBio L series Mouse Antibody Array L 308 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 28 1 Pa P ta P 2a P 2a psa P 3a Neg Neg 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 2 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 3 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 40 a 4 a2 42 4 43 43 44 44 45 45 46 46 47 47 48 48 49 49 50 50 51 51 52 52 53 53 54 54 55 55 56 56 5 57 57 58 58 59 59 60 60 61 61 62 62 63 63 64 64 65 65 66 66 67 67 68 68 69 69 70 70 6 71 71 72 72 73 73 74 74 75 75 76 76 77 77 78 78 79 79 80 80 81 81 82 82 83 83 84 84 7 85 85 86 86 87 87 88 88 89 89 go 90 91 91 92 92 93 93 94 94 95 95 96 96 97 97 98 98 8 99 99 100 100 101 101 102 102 103 103 104 104 105 105 106 106 107 107 108 108 109 109 110 110 141 111 112 112 9 n3 113 114 114 115 115 116 116 117 117 118 118 119 119 120 120 121 121 122 122 123 123 124
13. 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 You can calculate the normalized values as follows X Ny X y P1 P y Where P1 mean signal intensity of POS spots on reference array RavBio L Series Mouse Antibody Array L 308 Protocol 22 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 RavBio Analysis Tool software is available for use with data obtained using RayBio Biotin Label based Antibody Arrays 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 expression 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 in expression levels of each protein 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 m
14. atants e Seed cells at a density of 1x10 cells in 100 mm tissue culture dishes e Culture in complete culture medium for 24 48 hours e Replenish with serum free or low serum medium such as 0 2 FCS FBS serum and then incubate cells again for 48 hours t Recommended using membrane based array if using high serum medium such as 10 FCS FBS the glass slide arrays tend to have extremely high background for high serum containing media samples e Tocollect supernatants centrifuge at 1 000 g for 10 min and store as lt 1 ml aliquots at 80 C until needed RayBio L Series Mouse Antibody Array L 308 Protocol 4 Measure the total wet weight of cultured cells in the pellet and or culture dish Vou mav then normalize between arrays by dividing fluorescent signals by total cell mass i e express results as the relative amount of protein expressed mg total cell mass Or you can normalize between array by determining cell lylate concentration using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 Note The density of cells per dish used is dependent on the cell type More or less cells may be required Optimal culture time may vary and will depend on the cell line treatment conditions and other factors Bovine serum proteins produce detectable signals on the RayBio L Series Mouse Antibody Array 308 in media containing serum concentrations as low as 0 2 When testing serum containing med
15. ber with the plastic adhesive strips Note Avoid exposure to light in Steps 19 25 by covering the Glass Slide Assembly with aluminum foil or incubate in dark room 18 Incubate with Cy3 Conjugated Streptavidin at RT for 2 hours with gentle rocking or shaking Note Incubation may be done overnight at 4 C 19 Decant the solution and disassemble the glass slide from the incubation frame and chamber Disassemble the device by pushing clips outward from the side 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 gt Gamay the same side as the barcode ZS A 20 Gentiv place the glass slide into 30 ml Centrifuge Tube Item M Add enough 1X Wash Buffer to cover the entire glass slide Wash with gentle rocking or shaking for 10 min Remove the wash buffer Repeat 2 times for a total of 3 washes 21 Repeat step 20 this time with 1X Wash Buffer Il Repeat one time for a total of two washes for 5 min per wash RavBio L Series Mouse Antibody Array L 308 Protocol 15 22 Finallv wash the glass slide with 30 ml of de ionized or distilled water for 5 min Remove glass slide and decant water from Centrifuge Tube 23 Remove excess liquid from Centrifuge Tube and place glass slide into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets Make sure the finished glass slide is completelv drv before scanni
16. code CT l 2 printed sub arravs per glass chip Incubations and Washes Cover incubation chamber with a Plastic Adhesive Strip Item J to prevent evaporation during incubation or wash steps particularly those lasting 2 hours or longer During incubation and wash steps avoid foaming and be sure to remove all bubbles from the sub array surface Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle sec Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4 C 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 Unlike most Cy3 fluors the HiLyte Plus 532 used in this kit is very stable at RT and resistant to photobleaching on the hybridized glass slides However please protect glass slides from directly strong light and temperatures above RT RavBio L Series Mouse Antibodv Arrav L 308 Protocol 8 IV Protocol Assav Diagram 1 Cell culture supernatants 2 Serum or plasma or cell tissue lysates ATTN E 1 Preparation of sample 1 Serum sample 2 Dialysis of sample 2 Dialysis of sample 3 Determination of protein concentration 3 Biotinylation of sample 4 Biotinylation of sample i 4 Dialysis of biotinylated 5 Dialysis of biotinylated U sample 6 proceed to mic
17. diluted 2 fold with deionized or distilled water e Homogenize the tissue according to homogenizer manufacturer instructions e Transfer extracts to microcentrifuge tubes and centrifuge for 20 min at 13 000 rpm 4 C e Transfer supernatant to a clean tube and store at 70 C Note If the supernatant appears to be cloudy transfer the RayBio L Series Mouse Antibody Array L 308 Protocol supernatants to a clean tube centrifuge again at 13 000 rpm for 20 minutes at 2 8 C If the supernatant is still not clear store the lysate at 70 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C B Handling the glass slides e The microarrav slides are delicate Please do not touch the arrav surface with pipette tips forceps or vour fingers Hold the slides bv the edges oniv e Handle the slides with powder free gloves and in a clean environment e Do not remove the glass slide from the chamber assembly until step 19 and take great care not to break the glass slide when doing so 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 RavBio L Series Mouse Antibodv Arrav L 308 Protocol H C Lavout of Mouse L 308 Glass Slide D Two identical sub arravs on one slide 25mm em s 16mm Sub arrav 1 22mm 75mm Sub array 2 22mm Bar
18. easurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 RavBio L Series Mouse Antibody Array L 308 Protocol 23 VII Troubleshooting Guide Problem Cause Recommendation Weak signal Inadequate detection Check laser power and PMT parameters Inadequate reagent volumes Check pipettors and or improper dilution ensure correct preparation Short incubation times Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration Don t make too low dilution in sample Or concentrate sample Improper storage of kit Store kit at suggested temperature Higthbacksround Use more diluted sample Sample is too concentrated Excess of streptavidin Make sure to use the correct amount of streptavidin Inadequate detection Check laser power and PMT parameters Inadequate wash Increase the volume of wash buffer and incubation time Uneven signal Bubbles formed during incubation Avoid bubble formation during incubation Arrays are not completely covered by reagent Completely cover arrays with solution RayBio L Series Mouse Antibody Array L 308 Protocol 24 VIII Selected References 1 Christina Scheel et all Paracrine and Autocrine Signals Induce and Maintain Mesenchvmal and Stem Cell States in the Breast Cell 2011 145
19. ee VIII Selected References sse eennnenennznnni RavBio L Series Mouse Antibodv Arrav L 308 Protocol I Introduction Recent technological advances bv RavBiotech have enabled the largest commercially available antibody array to date With the L Series Antibodv Arrav 308 researchers can now obtain a broad panoramic view of cvtokine expression The expression levels of 308 mouse target proteins can be simultaneously detected including cytokines chemokines adipokine growth factors angiogenic factors proteases soluble receptors soluble adhesion molecules and other proteins in cell culture supernatants serum and plasma The first step in using the RayBio L Series Mouse Antibody Array 308 is to biotinylate the primary amine of the proteins in serum or plasma samples cell culture supernatant cell lysate or tissue lysate The glass slide arrays are then blocked just like a Western blot and the biotin labeled sample is added onto the glass slide which is pre printed with capture antibodies and incubated to allow for interaction of target proteins Streptavidin conjugated fluorescent dye Cy3 equivalent is then applied to the array Finally the glass slide is dried and laser fluorescence scanning is used to visualize the signals RavBio L Series Mouse Antibodv Arrav L 308 Protocol 2 Il Materials Provided A Storage Recommendations Upon receipt the kit should be stored at 20 C until needed Please use withi
20. ernatants or cell tissue lysate after dialysis procedure Step 3 We recommended using a BCA total protein assay eg Pierce Catalog 23227 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 RavBio L Series Mouse Antibody Array L 308 Protocol 10 4 Immediatelv before use prepare 1X Labeling Reagent Brieflv spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the tube pipette up and down or vortex slightiv to dissolve the Ivophilized 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 180 ul dialyzed sample 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 Note To normalize serum plasma concentrations during biotinylation measure sample volume before and after dialysis Then adjust the volumes of dialyzed serum plasma and Labeling Buffer to compensate to keep same total protein amount and total volume For example if serum plasma
21. ia we strongly recommend testing an uncultured media blank for comparison with sample results 2 Extracting Protein from Cells For attached cells remove supernatant from cell culture wash cells twice with cold 1X PBS For suspension cells pellet the cells by centrifuging using a microcentrifuge at 1500 rpm for 10 min Make sure to remove any remaining PBS before adding 1X RayBio L Series Mouse Antibody Array L 308 Protocol 3 Cell Lvsis Buffer 2X Cell Lvsis Buffer should be diluted 2 fold with deionized or distilled water Solubilize the cells at 2x10 cells ml in 1X Cell Lvsis Buffer e Pipette up and down to resuspend cells and rock the Ivsates gently at 2 8 C for 30 minutes Transfer extracts to microfugetubes and centrifuge at 13 000 rpm for 10 min at 2 8 C e Transfer supernatant to a clean tube Determining cell Ivlate concentration using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 Aliquot the lysates and store at 70 C Note If the supernatant appears to be cloudy transfer the supernatants to a clean tube centrifuge again at 13 000 rom for 20 minutes at 2 8 C If the supernatant is still not clear store the lysate at 70 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 3 Extracting Protein from Crude Tissue e Transfer approximate 100 mg crude tissue into a tube with 1 ml 1X Cell Lysis Buffer 2X Cell Lysis Buffer should be
22. ibody Array L 308 Protocol 19 VI Interpretation of Results A Explanation of Controls Spots 1 2 Positive Control spots POS1 POS2 POS3 are standardized amounts of biotinvlated 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 fluorescence 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 antibodies 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 B Typical results obtained with RayBio L Series Mouse Antibody Array L 308 The following figure shows the RavBio L Series Mouse Antibody Array 308 probed with serum sample The images were captured using a Axon GenePix laser scanner The strong signals in row 20 and the upper left and lower right corners of each RayBio L Series Mouse Antibody Array L 308 Protocol 20 arrav are Positive Controls which can be used to identifv the orientation and help normalize the results between arravs RavBio L Series Mouse Antibodv Arrav 308 Sample 1
23. io L Series Mouse Antibody Array L 308 Protocol 10 Block sub arravs bv adding 400 ul of Blocking Buffer Item F into each well of Assembled Glass Slide and incubating at RT for 30 min Ensure there are no bubbles on the arrav surfaces 11 Immediatelv prior to sample incubation spin biotin labeled samples for 5 min at 10 000 rpm to remove anv particulates or precipitants Dilute samples with Blocking Butter 3 Note Recommended dilution of the biotin labeled samples with Blocking Buffer prior to incubation is 2 10 fold for cell culture supernatants 20 fold for serum plasma or 30 fold cell tissue lysate Note Optimal sample dilution factor will depend on the abundance of target proteins If the background or antigen specific antibody signals are too strong the sample can be diluted further in subsequent experiments If the signal is too weak more concentrated samples can be used 12 Completely remove Blocking Buffer from each well Add 400 ul of diluted samples into appropriate wells Remove any bubbles on array surfaces Incubate arrays with gentle rocking or shaking for 2 hours at RT or overnight at 4 C Note Avoid the flow of sample into neighboring wells 13 Dilute 20X Wash Buffer Concentrate Item G 20 fold with de ionized or distilled water Decant the samples from each RayBio L Series Mouse Antibody Array L 308 Protocol well and wash 3 times with 800 ul of 1X Wash Buffer at RT with gentle rocking or shaking
24. n 6 months from the date of shipment After initial use remaining reagents should be stored at 4 C to avoid repeated freeze thaw cycles may be stored for up to 3 months Labeling Reagent Item B should be fresh preparation before use Unused glass slides should be kept at 20 C and avoid repeated freeze thaw cycles may be stored for up to 6 months RayBio L Series Mouse Antibody Array 308 ITEM DESCRIPTION Cat AAM BLG 1 2 Cat AAM BLG 1 4 TA Dialysis Vials ZE Labeling Reagent D StopSolution sss Solution 1 vial 50 ul RayBio L series Human Antibody 1L i 2 L li ep L 308 Glass Slides ki bu Sop Slides O F a Buffer 1 ma 8 m 1 noite 8 a H f20x Wash Butter batts 30m bate 30 mi TFT Cy8 Conjugated Streptaviin 1 Jk 2ves ees E f tube Each slide contains 2 identical subarravs HiLyte Plus 532 Only needed if testing cell or tissue lysates RayBio L Series Mouse Antibody Array L 308 Protocol 3 B Additional Materials Required e Distilled or de ionized water e KCI NaCl KH PO and Na HPO e Small plastic or glass containers e Orbital shaker or oscillating rocker e Beaker stir plate and stir bar e 1mltube e Pipettors pipette tips and other common lab consumables e Laser scanner for fluorescence detection list available online e Aluminum foil III Overview and General Considerations A Preparation and Storage of Samples 1 Preparation of Cell Culture Supern
25. ng or storage Note Alternativelv vou mav gentiv drv the glass slide using a low velocitv Nitrogen gas stream or ambiently in a laminar flow hood or similar clean environment Be sure to protect from light E Fluorescence Detection 24 You may proceed immediately to scanning or you may store the slide at 20 C in the Centrifuge Tube provided or at RT and to scan at a later time Note Unlike most Cy3 fluors the HiLyte Plus Fluor 532 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 RavBio L Series Mouse Antibody Array L 308 Protocol 16 into the incubation chamber by following step as shown in the figures below To avoid breaking the printed glass slide vou mav first want to practice assembling the device with a blank glass slide 1 rA Apply slide to incubation chamber barcode facing upward as in image A below Gently snap one edge of a snap on side as shown in image B Gently press other of side against lab bench and push in lengthwise direction image C Repeat with the other side image D RavBio L Series Mouse Antibodv Arrav L 308 Protocol 17 V Antibodv Arrav M
26. r like state J Cell Biochem 2011 112 5 1277 1285 10 Toh HC Wang W W Chia WK et al Clinical Benefit of Allogenic Melanoma Cell Lysate Pulsed Autologous Dendritic Cell Vaccine in MAGE Positive Colorectal Cancer Patients Clin Chem Res 2009 15 7726 7736 11 Zhen Hou Cytokine array analysis of peritoneal fluid between women with endometriosis of different stages and those withoutendometriosi Biomarkers 2009 14 8 604 618 12 Yao Liang Tang et al Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Circ Res 2009 109 197723 RavBio L Series Mouse Antibody Array L 308 Protocol 26 RavBio L series Antibodv Arravs are patent pending technologv developed bv RavBiotech This product is intended for research only and is not to be used for clinical diagnosis Our produces mav not be resold modified for resale or used to manufacture commercial products without written approval bv RavBiotech Inc Under no circumstances shall RavBiotech be liable for anv damages arising out of the use of the materials Products are guaranteed for six months from the date of shipment 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 RayBio is aregistered trademark of RayBiotech Inc
27. roarray Ss 5 proceed to microarrav AN analvsis S analvsis If using cell or tissue lysates start at step 2 Dialysis of sample A 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 KH PO and 3 45 g Na HPO in 2500 ml de ionized or distilled water Adjust pH 8 0 with 1M NaOH and adjust final volume to 3000 ml with de ionized or distilled water RavBio L Series Mouse Antibodv Arrav L 308 Protocol 9 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 172 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 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 h at 4C 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 protein concentration for cell culture sup
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