AAH BLM 1

RayBio Label-based (L-Series) Human Antibody Array L-507 or L-403 Membrane Kit Patent Pending Technology User Manual (Revised Feb 27, 2014) For the simultaneous detection of the relative expression of 507 (L-507) or 493 (L-493) human proteins in cell culture supernatants. L-Series Human Antibody Array L-507 Cat# AAH-BLM-1-2 (2 Sample Kit) Cat# AAH-BLM-1-4 (4 Sample Kit) L-Series Human Antibody Array L-493 Cat# AAH-BLM-2-2 (2 Sample Kit) Cat# AAH-BLM-2-4 (4 Sample Kit)

Please read manual carefully before starting experiment

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: [email protected]

RayBiotech, Inc TABLE OF CONTENTS I.

Introduction……..……………………………..........

2

How It Works………………..…………………………

3

Materials Provided………………………………….. A. Storage Recommendations…………….…… B. Additional Materials Required……………. III. Overview and General Considerations……..

3 3 5 6

A. Handling Array Membranes…… …………..

6

B. Incubation of Antibody Array ………………

6

IV. Protocol…………………………………………………….

7

A. Preparation of Samples…………………….....

8

B. Dialysis of Sample ……………………….……....

9

C. Biotin-labeling Sample …………………….....

10

D. Blocking and Incubation…………………….…

12

E. Detection………………….............................

13

Antibody Array Map…………………………….……

15

VI. Interpretation of Results…………………………..

21

II.

V.

VII. Troubleshooting Guide……………………………... 22 VIII. Reference List…...……………………….…............ 23 1 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

I. Introduction Recent technological advances by RayBiotech have enabled the largest commercially available antibody array to date. With the RayBio® L-Series Human Antibody Array 507 or 493, researchers can now obtain a broad, panoramic view of cytokine expression. The expression levels of 507 or 493 human 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.

The first step in using the Human L-507 or L-493 is to biotinylate the primary amine of the proteins in the sample. The membrane arrays are then blocked, similar to a Western blot, and the biotin-labeled sample is added onto the membrane array which is pre-printed with capture antibodies and incubated to allow for interaction of target proteins. After incubation with HRPConjugated Streptavidin, the signals can be visualized by chemiluminescence.

2 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

II. Materials Provided A. Storage Recommendations Upon receipt, Box 1 should be stored at -20 °C and Box 2 and Box 4 should be stored at 4 °C. The kit must be used within 6 months from the date of shipment. After initial use, Blocking Buffer, Stop Solution, HRP-Conjugated Streptavidin, Detection Buffers C and D should be stored at 4 °C to avoid repeated freezethaw cycles (may be stored for up to 3 months, Labeling Reagent, 3 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Item B should be fresh preparation before use). The Array Membrane should be kept at -20 °C and avoid repeated freezethaw cycles (may be stored for up to 6 months). 1. RayBio® L-Series Human Antibody Array 507 Box 1 (store at -20 °C): ITEM B D

DESCRIPTION Labeling Reagent Stop Solution

E

RayBio® L-Series Human 507 Antibody Array Membranes

Blocking Buffer 500X HRP-Conjugated I Streptavidin Concentrate Detection Buffer C K Detection Buffer D L Other Kit Components: F

cat#: AAH-BLM-1-2 Cat#: AAH-BLM-1-4 1 vials 2 vials 1 vial (50 μl) 2 membranes 4 membranes L-507 L-507 2 vials (30 ml/ea)

4 vials (30 ml/ea)

1 vial (100 ul) 1 vial (10 ml) 1 vial (10 ml) Plastic Sheets

2 vials (100 ul/ea) 2 vials (10 ml/ea) 2 vials (10 ml/ea)

Box 2 (store at 4 °C): ITEM A G H J N/A M

DESCRIPTION Dialysis Vials 20X Wash Buffer 1 Concentrate 20X Wash Buffer 2 Concentrate Spin Columns Plastic Incubation Trays (w/lid) Floating Dialysis Rack

Cat#: AAH-BLM-1-2 2 vials 1 vial (30 ml) 1 vial (30 ml) 2 columns 2 trays

Cat#: AAH-BLM-1-4 4 vials 1 vials (30 ml/ea) 1 vials (30 ml/ea) 4 columns 4 trays 1 rack

4 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

2. RayBio® L-Series Human Antibody Array 493 Box 1 (store at -20 °C): ITEM B D

DESCRIPTION Labeling Reagent Stop Solution RayBio® L-Series Human 493 Antibody Array Membranes

E

Blocking Buffer 500X HRP-Conjugated I Streptavidin Concentrate Detection Buffer C K Detection Buffer D L Other Kit Components: F

Cay#: AAH-BLM-2-2 Cat#: AAH-BLM-2-4 1 vials 2 vials 1 vial (50 μl) 2 membranes 4 membranes L-493 L-493 2 vials (30 ml/ea) 1 vial (100 ul) 1 vial (10 ml) 1 vial (10 ml)

4 vials (30 ml/ea) 2 vials (100 ul/ea) 2 vials (10 ml/ea) 2 vials (10 ml/ea) Plastic Sheets

Box 2 (store at 4 °C): ITEM A G H J N/A M

DESCRIPTION Dialysis Vials 20X Wash Buffer 1 Concentrate 20X Wash Buffer 2 Concentrate Spin Columns Plastic Incubation Trays (w/lid) Floating Dialysis Rack

Cat#: AAH-BLM-2-2 2 vials 1 vial (30 ml) 1 vial (30 ml) 2 columns 2 trays 1 rack

B. Additional Materials Required • • • • •

cat# AAH-BLM-2-4 4 vials 1 vials (30 ml/ea) 1 vials (30 ml/ea) 4 columns 4 trays

1X PBS, pH=8.0 Shaker 2~5 ml tube 50 ml conical collection tubes Distilled water 5

RayBio® L-Series Human Antibody Array 507 or 493 Protocol

• Kodak X-Omat™ AR film (REF 165 1454) and film processor or Chemiluminescence imaging system • large beaker • stir plate • Eppendorf tube

III. Overview and General Considerations A. Handling Array Membranes • Always use forceps to handle membranes and grip the membranes by the edges only. • Never allow membranes to dry during the experiment. • Avoid touching membranes with hands or any sharp tools.

B. Incubation • Completely cover membranes with sample or buffer during incubation and cover Plastic Incubation Tray with lid to avoid drying. • Avoid foaming during incubation steps. • Perform all incubation and wash steps under gentle rotation. • Several incubation steps such as step 3 in page 10 (sample incubation) or step 7 in page 11 (HRP-Conjugated Streptavidin incubation) may be done at 4 °C for overnight.

IV. Protocol 6 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Layout of L-507 or L-493 Array Membrane 7 cm 7 CM

CM 99cm

30 columns 36rows rows 30 columnsxx 36

Assay Diagram

7 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

A. Preparation of Samples 1). Seed cells at a density of 1x106 cells in 100 mm tissue culture dishes.* 2) Culture in complete culture medium for ~24–48 hours.** 3) Replenish with serum-free or low-serum medium, such as 0.2% FCS/FBS, and then re-incubate cells for ~48 hours*** 4) Collect the cell culture supernatant and centrifuge at 1,000 g for 10 minutes and store in ≤1 ml aliquots at -80 °C until needed. 5) Measure the total wet weight of the cultured cells in the pellet and/or culture dish. 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). Normalization can also be done between arrays by determining the total protein concentration using a total protein assay (RayBiotech recommends the Pierce BCA Protein Assay Kit, cat# 23227). Note: * The density of cells per dish used is dependent on the cell type. More or less cells may be required but should be determined empirically. ** Optimal culture time may be different and depends on cell lines, treatment conditions, and other factors. ***Bovine serum proteins produce detectable signals on the RayBio® L-Series membrane arrays at concentrations as low as 0.2%. When testing serum-containing media, it is 8 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

recommended test an uncultured media blank sample for comparison with sample results.

B. Dialysis of Sample Note: Samples must be dialyzed prior to biotin-labeling (Steps 5–7). 1.

Prepare dialysis buffer (1X PBS) by dissolving 0.6 g KCl, 24 g NaCl, 0.6 g KH2PO4 and 3.45 g Na2HPO4 in 2500 ml de-ionized or distilled water. Adjust to a pH of 8.0 with 1M NaOH and adjust final volume to 3000 ml with de-ionized or distilled water.

2.

Load each sample into a Dialysis Vials (Item A), 2.5-3.0 ml of sample per vial for dialyzing. Carefully place all Dialysis Vials into the Floating Rack.

3.

Place the Floating Rack into ≥500 ml dialysis buffer in a large beaker. Place beaker on a stir plate and dialyze for at least 3 hours at 4 °C, occasionally gently stirring the dialysis buffer. Then exchange the dialysis buffer with fresh buffer and repeat dialysis for at least 3 hours at 4 °C. Transfer dialyzed samples into a clean eppendorf tube. Centrifuge dialyzed samples for 5 minutes at 10,000 rpm to remove any particulates or precipitates and then transfer and combine each sample into one clean eppendorf tube. Mix well by gently pipetting.

9 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Note: The sample volume may change during dialysis. Note: Dialysis procedure may proceed overnight.

C. Biotin-labeling of Sample Avoid contamination with any solution containing amines (i.e., Tris, glycine) as well as azides during the biotinylation process. 4. Immediately before use, prepare 1X Labeling Reagent by briefly centrifuging down the Labeling Reagent vial (Item B) and add 100 µl 1X PBS (pH=8.0) into the vial. Pipette up and down or vortex briefly to dissolve the powder. 5. Add an appropriate amount* of 1X Labeling Reagent into the tube containing the sample and immediately mix the reaction solution. Incubate the reaction solution at room temperature for 30 minutes with gentle shaking. Gently tap the tube to mix the reaction solution every 5 minutes. * Use 7.2 µl of 1X Labeling Reagent for labeling 1 mg of total protein in samples. For example, if sample’s total protein concentration is 0.5 mg/ml you need to add 10.8 µl 1X Labeling Reagent to 3 ml dialyzed sample. Note: The total protein concentration needs to be determined if the sample volume changes after dialysis or if the total protein concentration was determined before the dialysis step. 10 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

6. Add 5 µl Stop Solution (Item D) into the reaction solution and then use the Spin Column (Item J) to remove any unbound biotin. a). Twist off the bottom closure of the Spin Column and loosen the cap (but keep the cap on). Place the Spin Column into a 50 ml conical collection tube. b). Centrifuge the Spin Column at 1,000 g for 3 minutes to remove storage solution. Note: The resin should appear compacted after centrifugation. c). Add 5 ml 1X PBS (pH=8.0) into the Spin Column and centrifuge at 1,000 x g for 3 minutes to remove the 1X PBS. Repeat an additional 2 times to wash the Spin Column. d). Place the Spin Column in a new 50 ml conical collection tube and slowly load 3.5 ml of sample to the center of the compact resin bed. Note: The maximal sample volume is 4 ml for each Spin Column. Do not load over 4 ml of sample into a Spin Column. e). Centrifuge the Spin Column at 1,000 x g for 3 minutes. The sample should filter through the resin and deposit into the 50 ml conical collection tube. Store at -80 0C until needed. Discard the Spin Column after use. 11 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

D. Blocking and Incubation 7. Place each membrane printed side up into a Plastic Incubation Tray (provided). 1 membrane per tray. Note: The printed membrane will have a “1” mark (for all L-507 membranes) “2” (for all L-493 membranes) in the upper left corner of the membrane. 8. Add 8 ml of Blocking Buffer (Item F) to each membrane and cover with the lid. Incubate at room temperature with gentle shaking for 1 hour. 9. Aspirate Blocking Buffer from each tray. Add 8 ml of diluted* or undiluted sample onto each membrane and cover with the lid. Incubate at room temperature with gentle shaking for 2 hours. Note: 1). It is recommended to use 8 ml of 5-fold diluted biotinlabeled cell culture supernatant. Dilute sample using Blocking Buffer. Note: 2). The concentration of sample used depends on the abundance of proteins. The samples can be concentrated if the overall signals are too weak. If the overall signals are too strong, the sample can be diluted further. Note: 3). Incubation may be done at room temperature with gentle shaking for 2 hours or overnight at 4°C. 12 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

10. Dilute 20X Wash Buffer 1 with deionized or distilled water to prepare the 1X Wash Buffer 1. Aspirate the samples from each tray and then wash by adding 20 ml of 1X Wash Buffer I at room temperature with gentle shaking (5 min per wash). Repeat the wash 2 more times for a total of 3 washes. 11. Aspirate the 1X Wash Buffer 1 from each tray. Dilute 20X Wash Buffer 2 with deionized or distilled water to prepare the 1X Wash Buffer 2. Wash 3 times with 20 ml of 1X Wash Buffer 2 at room temperature with gentle shaking. 12. Aspirate the 1X Wash Buffer 2 from each tray. Dilute the 500X HRP-Conjugated Streptavidin with Blocking Buffer to prepare the 1X HRP-Conjugated Streptavidin. Add 8 ml of 1X HRPConjugated Streptavidin to each membrane. Note: Ensure that the vial containing the 500X HRP-Conjugated Streptavidin is mixed well before use, as precipitation can form during storage. 13. Incubate at room temperature with gentle shaking for 2 hours. Note: incubation may be done at 4 0C for overnight. 14. Wash as directed in steps 10 and 11.

E. Detection 13 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

* Do not let the membrane dry out during detection. The detection process must be completed within 40 minutes without stopping. 15. For detection of 2 membranes, add 4.2 ml of Detection Buffer C and 4.2 ml of Detection buffer D into a tube and mix both solutions. Drain off excess wash buffer. Place membrane antibody side up (“1” or “2” symbol is marked in the top left corner of each membrane) on a clean plastic plate or its cover (provided in the kit). Pipette 4 ml of the mixed Detection Buffers on to each membrane and incubate at room temperature for 2 minutes with gentle shaking. Ensure that the detection mixture is evenly covering the membrane without any air bubbles. 16. Gently place the membrane with forceps (antibody side up) on a plastic sheet (provided) and cover the membrane with another plastic sheet. Gently smooth out any air bubbles. Avoid using pressure on the membrane. Work as quickly as possible. 17. The signal can be detected directly from the membrane using a chemiluminescence imaging system or by exposing the array to x-ray film (we recommend using Kodak X-Omat™ AR film) with subsequent development. Expose the membranes for 40 seconds. Then re-expose the film according to the intensity of signals. If the signals are too strong (background too high), reduce exposure time (eg, 5–30 seconds). If the signals are too weak, increase exposure time (eg, 5–20 min or overnight). Or re-incubate membranes overnight with 1X HRP-Conjugated 14 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Streptavidin, and repeat detection on the second day. 18. Save membranes at –20 °C to –80 °C for future reference.

V. Antibody Array Map RayBio® L-Series Human Antibody Array 507 or 493 Maps – if needed, larger versions of these maps can obtained by contacting technical support at 770-729-2992 or [email protected]. RayBio® L-Series Human Antibody Array 507 (L-507) Map P-1a

P-2a P-3a

27

28

29

30

P-1a N N 61 61 91 91 121 121 151 151 181 181 211 211 241 241 B B B B B B 361 361 391 391 421 421 451 451 B B

P-2a P-3a N 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 N N 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 N N 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 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 87 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 87 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 B B B 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 B B B 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 B B B 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 B B B 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 B B B 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 B B B 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 B B 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 B B 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507

28 58 58 88 88 118 118 148 148 178 178 208 208 238 238 268 268 298 298 328 328 358 358 388 388 418 418 448 448 478 478 N N

29 59 59 89 89 119 119 149 149 179 179 209 209 239 239 269 269 299 299 329 329 359 359 389 389 419 419 449 449 479 479 N N

30 60 60 90 90 120 120 150 150 180 180 210 210 240 240 270 270 300 300 330 330 360 360 390 390 420 420 450 450 480 480 N N

B B

B B

B B

N

B B

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536

15 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

N N

P-3b P-2b P-1b P-3b P-2b P-1b

RayBio® L-Series Human Antibody Array 507 (L-507) List Number 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 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Name Positive 1a Positive 2a Positive 3a neg 6Ckine Activin A Activin B Activin C Activin RIA / ALK-2 Activin RIB / ALK-4 Activin RII A/B Activin RIIA Adiponectin / Acrp30 AgRP ALCAM Angiogenin Angiopoietin-1 Angiopoietin-2 Angiopoietin-4 Angiopoietin-like 1 Angiopoietin-like 2 Angiopoietin-like Factor Angiostatin APJ AR (Amphiregulin) APRIL Artemin Axl B7-1 /CD80 BAFF R / TNFRSF13C neg neg neg BCMA / TNFRSF17 BD-1 BDNF beta-Catenin BAX beta-NGF BIK BLC / BCA-1 / CXCL13 BMP-2 BMP-3 BMP-3b / GDF-10 BMP-4 BMP-5 BMP-6 BMP-7 BMP-8 BMP-15 BMPR-IA / ALK-3 BMPR-IB / ALK-6 BMPR-II BTC Cardiotrophin-1 / CT-1 CCL14 / HCC-1 / HCC-3 CCL28 / VIC CCR1 CCR2 CCR3

Number 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 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Name CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CD14 CD27 / TNFRSF7 CD30 / TNFRSF8 CD30 Ligand / TNFSF8 CD40 / TNFRSF5 CD40 Ligand / TNFSF5 /CD154 CD 163 Cerberus 1 Chem R23 Chordin-Like 1 Chordin-Like 2 Csk CLC CNTF CNTF R alpha Coagulation Factor III / Tissue Factor CRIM 1 Cripto-1 CRTH-2 Cryptic CTACK / CCL27 CTGF / CCN2 CTLA-4 /CD152 CV-2 / Crossveinless-2 CXCL14 / BRAK CXCL16 CXCR1 / IL-8 RA CXCR2 / IL-8 RB CXCR3 CXCR4 (fusin) CXCR5 /BLR-1 CXCR6 D6 DAN DANCE DcR3 / TNFRSF6B Decorin Dkk-1 Dkk-3 Dkk-4 DR3 / TNFRSF25 DR6 / TNFRSF21 Dtk EDA-A2 EDAR EDG-1 EGF EGF R / ErbB1 EG-VEGF / PK1 EMAP-II ENA-78 Endocan Endoglin / CD105 Endostatin

Number 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

Name EN-RAGE Eotaxin / CCL11 Eotaxin-2 / MPIF-2 Eotaxin-3 / CCL26 Epiregulin ErbB2 ErbB3 ErbB4 Erythropoietin E-Selectin Endothelin FADD FAM3B Fas / TNFRSF6 Fas Ligand FGF Basic FGF-BP FGF R3 FGF R4 FGF R5 FGF-4 FGF-5 FGF-6 FGF-7 / KGF FGF-8 FGF-9 FGF-10 / KGF-2 FGF-11 FGF-12 FGF-13 1B FGF-16 FGF-17 FGF-18 FGF-19 FGF-20 FGF-21 FGF-23 FLRG Flt-3 Ligand Follistatin Follistatin-like 1 Fractalkine Frizzled-1 Frizzled-3 Frizzled-4 Frizzled-5 Frizzled-6 Frizzled-7 Galectin-3 GASP-1 / WFIKKNRP GASP-2 / WFIKKN GCP-2 / CXCL6 GCSF G-CSF R / CD 114 GDF1 GDF3 GDF5 GDF8 GDF9 GDF11

16 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Number 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

Name GDF-15 GDNF GFR alpha-1 GFR alpha-2 GFR alpha-3 GFR alpha-4 GITR / TNFRF18 GITR Ligand / TNFSF18 Glucagon Glut1 Glut2 Glut3 Glut5 Glypican 3 Glypican 5 GM-CSF GM-CSF R alpha Granzyme A GREMLIN GRO GRO-a Growth Hormone (GH) Growth Hormone R (GHR) HB-EGF HCC-4 / CCL16 HCR / CRAM-A/B Hepassocin GLO-1 HGF HGFR HRG1-alpha / NRG1-alpha HRG1-beta1 / NRG1-beta1 HVEM / TNFRSF14 I-309 ICAM-1 ICAM-2 ICAM-3 (CD50) ICAM-5 IFN-alpha / beta R1 IFN-alpha / beta R2 IFN-beta IFN-gamma IFN-gamma R1 IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-6 IGFBP-rp1 / IGFBP-7 IGF-I IGF-I SR IGF-II IGF-II R IL-1 alpha IL-1 beta IL-1 F5 / FIL1delta IL-1 F6 / FIL1 epsilon IL-1 F7 / FIL1 zeta IL-1 F8 / FIL1 eta IL-1 F9 / IL-1 H1

Number 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

Name IL-1 F10 / IL-1HY2 IL-1 R3 / IL-1 R AcP IL-1 R4 /ST2 IL-1 R6 / IL-1 Rrp2 IL-1 R8 IL-1 R9 IL-1 ra IL-1 sRI IL-1 sRII IL-2 IL-2 R alpha IL-2 R beta /CD122 IL-2 R gamma IL-3 IL-3 R alpha IL-4 IL-4 R IL-5 IL-5 R alpha IL-6 IL-6 R IL-7 IL-7 R alpha IL-8 IL-9 IL-10 IL-10 R alpha IL-10 R beta IL-11 IL-12 p40 Blank Blank Blank Blank IL-12 p70 IL-12 R beta 1 IL-12 R beta 2 IL-13 IL-13 R alpha 1 IL-13 R alpha 2 IL-15 IL-15 R alpha IL-16 IL-17 IL-17B IL-17B R IL-17C IL-17D IL-17E IL-17F IL-17R IL-17RC IL-17RD IL-18 BPa IL-18 R alpha /IL-1 R5 IL-18 R beta /AcPL IL-19 IL-20 IL-20 R alpha IL-20 R beta

RayBio® L-Series Human Antibody Array 507 (L-507) List continued Number 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

Name Blank Blank Blank Blank IL-21 IL-21 R IL-22 IL-22 BP IL-22 R IL-23 IL-23 R IL-24 IL-26 IL-27 IL-28A IL-29 IL-31 IL-31 RA BACE-1 FACX Insulin Insulin R Insulysin / IDE IP-10 I-TAC / CXCL11 Kininostatin / kininogen Kremen-1 Kremen-2 Lck Latent TGF-beta bp1 Blank Blank Blank Blank LBP LECT2 Lefty - A Leptin R Leptin (OB) LFA-1 alpha LIF LIF R alpha LIGHT / TNFSF14 Lipocalin-1 LRP-1 LRP-6 L-Selectin (CD62L) Lipocalin-2 Lymphotactin / XCL1 Lymphotoxin beta / TNFSF3 Lymphotoxin beta R / TNFRSF3 MAC-1 MCP-1 MCP-2 MCP-3 MCP-4 / CCL13 M-CSF M-CSF R MDC MFG-E8

Number 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

Name MFRP MIF MIG MIP-1a MIP-1b MIP-1d MIP 2 MIP-3 alpha MIP-3 beta MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 /Stromelysin-3 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 / MT3-MMP MMP-19 MMP-20 MMP-24 / MT5-MMP MMP-25 / MT6-MMP Musk MSP alpha Chain MICA NAP-2 NCAM-1 / CD56 Neuritin NeuroD1 Neuropilin-2 Neurturin NGF R NOV / CCN3 NRG1 Isoform GGF2 Nidogen-1 NrCam NRG2 NRG3 NT-3 NT-4 Orexin A Orexin B OSM Osteoactivin / GPNMB Osteocrin Osteoprotegerin / TNFRSF11B OX40 Ligand / TNFSF4 PARC / CCL18 PD-ECGF PDGF R alpha PDGF R beta PDGF-AA PDGF-AB PDGF-BB PDGF-C PDGF-D

Number 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

Name PECAM-1 /CD31 Pentraxin3 / TSG-14 Persephin PF4 / CXCL4 PlGF PLUNC Pref-1 Progranulin Prolactin P-selectin RAGE RANK / TNFRSF11A RANTES RELM beta RELT / TNFRSF19L ROBO4 S100 A8/A9 S100A10 SAA SCF SCF R /CD117 SDF-1 / CXCL12 sFRP-1 sFRP-3 sFRP-4 sgp130 SIGIRR Siglec-5/CD170 Siglec-9 SLPI Smad 1 Smad 4 Smad 5 Smad 7 Smad 8 Prdx6 Soggy-1 Sonic Hedgehog (Shh N-terminal) SPARC Spinesin TACI / TNFRSF13B Tarc TCCR / WSX-1 TECK / CCL25 TFPI TGF-alpha TGF-beta 1 TGF-beta 2 TGF-beta 3 TGF-beta 5 TGF-beta RI / ALK-5 TGF-beta RII Grb2 TGF-beta RIII Thrombopoietin (TPO) TPX Thrombospondin-1 Thrombospondin-2 Thrombospondin-4 Thymopoietin

17 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Number 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

Name Blank Blank Blank Tie-1 Tie-2 TIMP-1 TIMP-2 TIMP-3 TIMP-4 TL1A / TNFSF15 TLR1 TLR2 TLR3 TLR4 TMEFF1 / Tomoregulin-1 TMEFF2 TNF-alpha TNF-beta TNF RI / TNFRSF1A TNF RII / TNFRSF1B TRADD TRAIL / TNFSF10 TRAIL R1 / DR4 / TNFRSF10A TRAIL R2 / DR5 / TNFRSF10B TRAIL R3 / TNFRSF10C TRAIL R4 / TNFRSF10D TRANCE Neg Neg Neg Positive 2a Positive 2b Positive 2c Blank TREM-1 TROY / TNFRSF19 TSG-6 TSLP R TWEAK / TNFSF12 TWEAK R / TNFRSF12 Ubiquitin+1 uPA uPAR Vasorin VCAM-1 (CD106) VE-Cadherin VEGF VEGF R2 (KDR) VEGF R3 VEGF-B VEGF-C VEGF-D VEGI / TNFSF15 WIF-1 WISP-1 / CCN4 XEDAR Neg Positive 3b Positive 2b Positive 1b

RayBio® L-Series Human Antibody Array 493 (L-493) Map P-1a

P-2a

P-3a

N

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

P-1a N N 61 61 91 91 121 121 151 151 181 181 211 211 241 241 B B B B B B 361 361 391 391 421 421 451 451 B B

P-2a N N 62 62 92 92 122 122 152 152 182 182 212 212 242 242 B B B B B B 362 362 392 392 422 422 452 452 B B

P-3a N N 63 63 93 93 123 123 153 153 183 183 213 213 243 243 B B B B B B 363 363 393 393 423 423 453 453 B B

N 34 34 64 64 94 94 124 124 154 154 184 184 214 214 244 244 B B B B B B 364 364 394 394 424 424 454 454 484 484

5 35 35 65 65 95 95 125 125 155 155 185 185 215 215 245 245 275 275 305 305 335 335 365 365 395 395 425 425 455 455 485 485

6 36 36 66 66 96 96 126 126 156 156 186 186 216 216 246 246 276 276 306 306 336 336 366 366 396 396 426 426 456 456 486 486

7 37 37 67 67 97 97 127 127 157 157 187 187 217 217 247 247 277 277 307 307 337 337 367 367 397 397 427 427 457 457 487 487

8 38 38 68 68 98 98 128 128 158 158 188 188 218 218 248 248 278 278 308 308 338 338 368 368 398 398 428 428 458 458 488 488

9 39 39 69 69 99 99 129 129 159 159 189 189 219 219 249 249 279 279 309 309 339 339 369 369 399 399 429 429 459 459 489 489

10 40 40 70 70 100 100 130 130 160 160 190 190 220 220 250 250 280 280 310 310 340 340 370 370 400 400 430 430 460 460 490 490

11 41 41 71 71 101 101 131 131 161 161 191 191 221 221 251 251 281 281 311 311 341 341 371 371 401 401 431 431 461 461 491 491

12 42 42 72 72 102 102 132 132 162 162 192 192 222 222 252 252 282 282 312 312 342 342 372 372 402 402 432 432 462 462 492 492

13 43 43 73 73 103 103 133 133 163 163 193 193 223 223 253 253 283 283 313 313 343 343 373 373 403 403 433 433 463 463 493 493

14 44 44 74 74 104 104 134 134 164 164 194 194 224 224 254 254 284 284 314 314 344 344 374 374 404 404 434 434 464 464 494 494

15 45 45 75 75 105 105 135 135 165 165 195 195 225 225 255 255 285 285 315 315 345 345 375 375 405 405 435 435 465 465 495 495

16 46 46 76 76 106 106 136 136 166 166 196 196 226 226 256 256 286 286 316 316 346 346 376 376 406 406 436 436 466 466 496 496

17 47 47 77 77 107 107 137 137 167 167 197 197 227 227 257 257 287 287 317 317 347 347 377 377 407 407 437 437 467 467 497 497

18 48 48 78 78 108 108 138 138 168 168 198 198 228 228 258 258 288 288 318 318 348 348 378 378 408 408 438 438 468 468 498 498

19 49 49 79 79 109 109 139 139 169 169 199 199 229 229 259 259 289 289 319 319 349 349 379 379 409 409 439 439 469 469 499 499

20 50 50 80 80 110 110 140 140 170 170 200 200 230 230 260 260 290 290 320 320 350 350 380 380 410 410 440 440 470 470 500 500

21 51 51 81 81 111 111 141 141 171 171 201 201 231 231 261 261 291 291 321 321 351 351 381 381 411 411 441 441 471 471 501 501

22 52 52 82 82 112 112 142 142 172 172 202 202 232 232 262 262 292 292 322 322 352 352 382 382 412 412 442 442 472 472 502 502

23 53 53 83 83 113 113 143 143 173 173 203 203 233 233 263 263 293 293 323 323 353 353 383 383 413 413 443 443 473 473 503 503

24 54 54 84 84 114 114 144 144 174 174 204 204 234 234 264 264 294 294 324 324 354 354 384 384 414 414 444 444 474 474 504 504

25 55 55 85 85 115 115 145 145 175 175 205 205 235 235 265 265 295 295 325 325 355 355 385 385 415 415 445 445 475 475 505 505

26 56 56 86 86 116 116 146 146 176 176 206 206 236 236 266 266 296 296 326 326 356 356 386 386 416 416 446 446 476 476 506 506

27 57 57 87 87 117 117 147 147 177 177 207 207 237 237 267 267 297 297 327 327 357 357 387 387 417 417 447 447 477 477 507 507

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

B B

N N

515 516 517 518 519 520 521 522 515 516 517 518 519 520 521 522

18 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

28

29

28 29 58 59 58 59 88 89 88 89 118 119 118 119 148 149 148 149 178 179 178 179 208 209 208 209 238 239 238 239 268 269 268 269 298 299 298 299 328 329 328 329 358 359 358 359 388 389 388 389 418 419 418 419 448 449 448 449 478 479 478 479 N N N N

30 30 60 60 90 90 120 120 150 150 180 180 210 210 240 240 270 270 300 300 330 330 360 360 390 390 420 420 450 450 480 480 N N

P-3b P-2b P-1b P-3b P-2b P-1b

RayBio® L-Series Human Antibody Array 493 (L-493) List Number 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 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Name Positive 1a Positive 2a Positive 3a neg 11b-HSD1 2B4 4-1BB A1BG A2M ABL1 ACE ACE-2 ACK1 ACPP ACTH ADAM-9 ADAMTS-1 ADAMTS-10 ADAMTS-13 ADAMTS-15 ADAMTS-17 ADAMTS-18 ADAMTS-19 ADAMTS-4 ADAMTS-5 ADAMTS-L2 Adipsin Afamin AFP ALBUMIN neg neg neg Aldolase A Aldolase B Aldolase C ALK Alpha 1 AG Alpha 1 Microglobulin Alpha Lactalbumin ALPP AMICA AMPKa1 Amylin ANGPTL3 ANGPTL4 Annexin A7 APC APCS Apelin Apex1 APN ApoA1 ApoA2 ApoA4 ApoB ApoB100 ApoC1 ApoC2 ApoC3

Number 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 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

Name ApoD ApoE ApoE3 ApoH ApoM APP ASPH Attractin B3GNT1 BAF57 BAFF BAI-1 BCAM Beta 2M Beta Defensin 4 Beta IG-H3 Biglycan BLAME BMP-9 BMX BNIP2 BNP Btk C2 C3a C5/C5a C7 C8B C9 CA9 CA15-3 CA19-9 CA125 Cadherin-13 Calbindin Calbindin D Calcitonin Calreticulin Calsyntenin-1 CART Caspase-3 Caspase-8 Cathepsin B Cathepsin D Cathepsin L Cathepsin S CBP CCK CD23 CD24 CD36 CD38 CD44 CD45 CD46 CD47 CD55 CD59 CD61 CD71

Number 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

Name CD74 CD79 alpha CD90 CD97 CD200 CEA CEACAM-1 Ceruloplasmin CFHR2 Chemerin CHI3L1 Chromogranin A Chymase cIAP-2 Ck beta 8-1 CK-MB Claudin-3 Claudin-4 CLEC3B Clusterin CNDP1 COCO Complement factor H Contactin-1 Contactin-2 Corticosteroid-binding globulin COX-2 C-peptide CPN2 Creatinine CRP CRTAM CSH1 cTnT CutA Cyclin D1 Cystatin A Cystatin B Cystatin C Cytochrome C Cytokeratin 8 Cytokeratin18 Cytokeratin19 DBI DCBLD2 D-Dimer DEFA1/3 Defensin Desmin DLL1 DLL4 DMP-1 DPPIV E-Cadherin Endorphin Beta Endothelin Receptor A Enolase 2 ENPP2 EpCAM EphA1

19 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Number 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240

Name EphA2 EphA3 EphA4 EphA5 EphA6 EphA7 EphA8 EphB1 EphB2 EphB3 EphB4 EphB6 ERRa Erythropoietin R ESAM EV15L EXTL2 FABP1 FABP2 FABP3 FABP4 Factor XIII A Factor XIII B FAK FAP Fc RIIB/C Fen 1 FER Ferritin Fetuin A Fetuin B FGFR1 FGFR1 alpha FGFR2 Fibrinogen Fibrinopeptide A Fibronectin Ficolin-3 FIH FOLR1 FOXN3 FoxO1 FoxP3 FRK FSH Furin Fyn GADD45A Galanin Galectin-1 Galectin-3BP Galectin-7 gamma-Thrombin Gas1 Gastrin GATA-3 GATA-4 Gelsolin Ghrelin GLP-1

Number 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

Name GMNN GPBB GPI GPR-39 GPX1 GPX3 GRP GRP75 GRP78 GSR GST HADHA HAI-1 HAI-2 Haptoglobin hCG alpha hCGb Hck HE4 Hemopexin Hepcidin HOXA10 HSP10 HSP20 HSP27 HSP32 HSP40 HSP60 HSP70 HSP90 Blank Blank Blank Blank HSPA8 HTRA2 IBSP IGF2BP1 IGFBP-5 IL-23p19 IL-33 IL-34 IL36RN INSL3 INSRR Integrin alpha V Itk ITM2B Kallikrein 2 Kallikrein 5 Kallikrein 6 Kallikrein 7 Kallikrein 8 Kallikrein 10 Kallikrein 11 Kallikrein 14 KCC3 KCTD10 KIF3B KLF4

RayBio® L-Series Human Antibody Array 493 (L-493) List continued Number 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360

Name Blank Blank Blank Blank LAG-3 Layilin LDL R Legumain LH LIMPII LIN41 Livin LOX-1 LPS LRG1 LTF LTK Lumican Lyn LYRIC LYVE-1 LZTS1 Mammaglobin A Marapsin MATK MBL MBL-2 Mer Mesothelin MICB Blank Blank Blank Blank Midkine MINA MSHa MTUS1 Myoglobin NAIP Nanog NELL2 NEP Nesfatin Nestin NET1 Netrin G2 Netrin-4 Neurokinin-A Neuropeptide Y NF1 NM23-H1/H2 Notch-1 NPTX1 NPTXR NR3C3 Ntn1 OCT3/4 Omentin Osteocalcin

Number 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

Name Osteopontin OX40 p21 p27 p53 PAI-1 PAK7 Pancreastatin Pancreatic Polypeptide Pappalysin-1 PARK7 P-Cadherin PCAF PD-1 PDX-1 PEDF PEPSINOGEN I PEPSINOGEN II PGRP-S PI 16 PI 3Kinase p85 beta PIM2 PKM2 Plasminogen Podocalyxin POMC PON1 PON2 PPARg2 PPP2R5C Presenilin 1 Presenilin 2 Pro-BDNF Procalcitonin Pro-Cathepsin B Progesterone pro-Glucagon Prohibitin Pro-MMP-7 Pro-MMP-9 Pro-MMP-13 ProSAAS Prostasin Protein p65 PSA-Free PSA-total PSP PTH PTHLP PTN PTPRD PYK2 PYY Ras RBP4 RECK RELM alpha Resistin RET RIP1

Number 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

Name ROCK1 ROCK2 ROR1 ROR2 ROS RYK S100A4 S100A6 S100A8 S-100b SART1 SART3 SCG3 Selenoprotein P SEMA3A Serotonin Serping 1 Serpin A1 Serpin A3 Serpin A4 Serpin A5 Serpin A8 Serpin A9 Serpin A12 Serpin B5 Serpin D1 Serpin I1 SERTAD2 SHBG SMAC SNCG Somatotropin SOST SOX17 SOX2 SPARCL1 SPINK1 SRMS SSEA-1 SSEA-4 SSTR2 SSTR5 Survivin SYK Syndecan-1 Syndecan-3 TACE TAF4 Tec TFF1 TFF3 Thrombin Thrombomodulin Thymidine Kinase-1 Thyroglobulin TIM-1 TNK1 TOPORS TPA TPM1

20 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Number 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

Name Blank Blank Blank TRA-1-60 TRA-1-81 Transferrin Trappin-2 TRKB Troponin I Troponin C TRPC1 TRPC6 TRPM7 Trypsin 1 TSH TSLP TXK Tyk2 TYRO10 Uromodulin Vasopressin VDUP-1 VEGF R1 VGF VIP Receptor 2 Visfatin Vitamin D Receptor Neg Neg Neg Blank Blank Blank Blank Vitamin D-BP Vitamin K-dependent protein S Vitronectin VWF Wilms Tumor 1 XIAP ZAG ZAP70 Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank Neg Positive 3b Positive 2b Positive 1b

VI. Interpretation of Results The following images show the RayBio® L-Series Human Antibody Array 1000 captured using a chemiluminescence imaging system (UVP BioImaging Systems). To obtain optimal results, it is suggested to try several different exposure times until the best one is determined. Then, by comparing the signal intensities, relative expression levels of the target proteins can be made. The intensities of signals can be quantified by densitometry. Anti-HRP (P-1a, P-2a, P-3a) and anti-streptavidin (P-1b, P-2b, P-3b) will produce positive control signals, which can be used to identify the orientation and help normalize the results from different arrays being compared. Antibody affinity to its target varies significantly between antibodies. The intensity detected on the array with each antibody depends on this affinity; therefore, signal intensity comparison can be performed only within the same antibody/antigen system and not between different antibodies. The RayBio® Analysis Tool is a program specifically designed for analysis of RayBio® L-Series Human Antibody Antibody Arrays. This tool will not only assist in compiling and organizing your data, but also reduces your calculations to a “copy and paste.” Call RayBiotech, Inc. at 770-729-2992 for ordering information.

21 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

L-507

L-493

22 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

VII. Troubleshooting Guide Problem

Cause

Weak signal or no 1. Taking too much time signal for detection. 2. Film developer does not work properly. 3. Did not mix HRPstreptavidin well before use. 4. Sample is too dilute.

5. Other.

Recommendation 1. The whole detection process must be completed in 30 min. 2. Fix film developer. 3. Mix tube containing HRP-Conjugate Streptavidin well before use since precipitates may form during storage. 4. Increase sample concentration 1.Check if there were any contamination with any solution containing amines in biotin-labeling step Tris, glycine)increase as well as azides during the biotinylation 2. Slightly HRP concentrations. 3. Work as quickly as possible after mix Detection Buffer C and D . 4. Expose film for overnight to detect weak signal.

Uneven signal

1. Bubbles formed during incubation.

2. Membranes were not completely covered by solution. High background 1. Exposure time is too long. 2. Membranes dry out during experiment. 3. Sample is too concentrated.

1. Remove bubbles during incubation.

2. Completely cover membranes with solution.

1. Decrease exposure time. 2. Completely cover membranes with solution during experiment. Cover tray w/ lid 3. Dilute sample.

23 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

VIII. Reference List 1. Christina Scheel et all. Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Cell. 2011;145, 926–940 2. Lin Y, Huang R, Chen L, et al. Profiling of cytokine expression by biotin-labeled-based protein arrays. Proteomics. 2003, 3: 1750–1757. 3. 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. 4. 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. 5. 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. 6. Jun Zhong et all. Temporal Profiling of the Secretome during Adipogenesis in Humans. Journal of Proteome Research. 2010, 9, 5228–5238 7. Chowdury UR, Madden BJ, Charlesworth MC, Fautsch MP. 24 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Proteomic Analysis of Human Aqueous Humor. Invest Ophthalmol Visual Sci. 2010; 51(10):4921–4931. 8. Wei Y, Cui C, Lainscak M, et al. Type-specific dysregulation of matrix metalloproteinases and their tissue inhibitors in endstage heart failure patients: relationshp between MMP-10 and LV remodeling. J Cell Mol Med. 2011; 15(4):773–782. 9. Kuranda K, Berthon C, Lepêtre F, et al. Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor-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 without endometriosi 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

25 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

RayBio® is the trademark of RayBiotech, Inc. This product is intended for research only and is not to be used for clinical diagnosis. These products may not be resold, modified for resale, or used for manufacture of commercial products without express 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 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.

26 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

Note:

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Note:

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Note:

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32 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

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This product is for research use only.

©2004 RayBiotech, Inc.

35 RayBio® L-Series Human Antibody Array 507 or 493 Protocol