AAH BLM 2B

RayBio Label-based (L-Series) Human Antibody Array L-507 or L-493 Membrane Kit Patent Pending Technology User Manual (R...
0 downloads 194 Views 917KB Size
DOWNLOAD .PDF
RayBio Label-based (L-Series) Human Antibody Array L-507 or L-493 Membrane Kit Patent Pending Technology User Manual (Revised January 25, 2016) For the simultaneous detection of the relative expression of 507 (L-507) or 493 (L-493) human proteins in tissue lysates and serum/plasma.

L-Series Human Antibody Array L-507: For Cell/Tissue and Serum/Plasma Cat# AAH-BLM-1B-2 (2 Sample Kit) Cat# AAH-BLM-1B-4 (4 Sample Kit) L-Series Human Antibody Array L-493 Cat# AAH-BLM-2B-2 (2 Sample Kit) Cat# AAH-BLM-2B-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 and How it Works ……….........

2

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

4 4 6 7

A. Preparation of Samples………………………..

7

B. Handling Array Membranes ……..…………

9

C. Incubation of Antibody Array………………..

9

II.

IV. Protocol………………………………………………….….

10

A. Dialysis of Sample ……………………….……...

11

B. Biotin-labeling Sample …………………….....

12

C. Blocking and Incubation…………..……….…

14

D. Detection…………………..............................

16

Antibody Array Map…………………………………

18

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

24

VII. Troubleshooting Guide…………………………....

26

VIII. Reference List…...………………………...….........

27

V.

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 target proteins can be simultaneously detected, including cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules and other proteins in serum, plasma, cell culture supernatants, and cell and tissue lysates.

The first step in using the Human L-507 or L-493 array is to biotinylate the primary amines 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. The membrane is then incubated to allow for interaction of target proteins. After incubation with HRP-Conjugated Streptavidin, the signals can be visualized by chemiluminescence.

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

3 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 should be stored at 4 °C. The kit must be used within 6 months from the date of shipment. After initial use, the Blocking Buffer, Stop Solution, HRP-Conjugated Streptavidin and Detection Buffers C and D 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 prepared fresh each time before use. The Array Membrane should be kept at -20 °C and repeated freeze-thaw cycles should be avoided (may be stored for up to 6 months).

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

RayBio® L-Series Human Antibody Array 507 Box 1 (store at -20 °C): ITEM B D

DESCRIPTION Labeling Reagent Stop Solution RayBio L-Series Human 507 E Antibody array membrane F Blocking Buffer 500X HRP-Conjugated I Streptavidin Concentrate K Detection Buffer C L Detection Buffer D Other Kit Components: Plastic Sheets

AAH-BLM-1B-2 AAH-BLM-1B-4 1 vial 2 vials 1 vial (50 µl) 2 membranes 4 membranes L-507 L-507 2 vials (30 ml/ea) 4 vials (30 ml/ea)

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

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

1 vial (100 µl)

2 vials (100 µl/ea)

1 vial (10 ml) 1 vial (10 ml)

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

AAH-BLM-1B-2 2 vials 1 vial (30 ml) 1 vial (30 ml) 2 columns 2 trays

AAH-BLM-1B-4 4 vials 1 vial (30 ml) 1 vial (30 ml) 4 columns 4 trays

1 rack 1 bottle (10 ml)

5 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 507 E Antibody array membrane F Blocking Buffer 500X HRP-Conjugated I Streptavidin Concentrate K Detection Buffer C L Detection Buffer D Other Kit Components: Plastic Sheets

AAH-BLM-2B-2 AAH-BLM-2B-4 1 vial 2 vials 1 vial (50 µl) 2 membranes 4 membranes L-493 L-493 2 vials (30 ml/ea) 4 vials (30 ml/ea)

Box 2 (store at 4 °C):

ITEM DESCRIPTION A-2 Dialysis Vials G 20X Wash Buffer 1 Concentrate H 20X Wash Buffer 2 Concentrate J-2 Spin Columns N/A Plastic Incubation Trays (w/lid) M Floating Dialysis Rack N/A 2X Lysis Buffer* * Only need if running cell or tissue lysates

1 vial (100 µl)

2 vials (100 µl/ea)

1 vial (10 ml) 1 vial (10 ml)

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

AAH-BLM-2B-2 2 vials 1 vial (30 ml) 1 vial (30 ml) 2 columns 2 trays

AAH-BLM-2B-4 4 vials 1 vial (30 ml) 1 vial (30 ml) 4 columns 4 trays

1 rack 1 bottle (10 ml)

B. Additional Materials Required • • • • •

1X PBS, pH=8.0 Shaker 2 - 5 ml tube 50 ml conical collection tubes Distilled water 6

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 tubes III. Overview and General Considerations A. Preparation and Storage of Samples 1. Extracting Protein from Cells 1)

Centrifuge cells. a. Adherent cells: i. Remove supernatant from the cell culture and wash cells gently two times with cold 1X PBS taking care not to disturb the cell layer. ii. Add enough cold 1X PBS to cover cell layer and use cell scraper to detach cells. Proceed to b. Cells in Suspension. b. Cells in Suspension: i. Pellet the cells by centrifuging using a microcentrifuge tube at 1500 rpm for 10 minutes.

2)

Make sure to remove any remaining PBS before adding 1X Cell Lysis Buffer (2X Cell Lysis Buffer should be diluted 2-fold with deionized or distilled water). Solubilize the cells at 2x107 cells/ml in 1X Cell Lysis Buffer. 7

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

3)

Pipette up and down to resuspend the cells, and rock the lysates gently at 2-8 °C for 30 minutes. Transfer the lysates to microfuge tubes and centrifuge at 13,000 rpm for 10 minutes at 2-8 °C. Note: If the supernatant appears to be cloudy, transfer the supernatant 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 -80°C for 20 minutes. Remove from the freezer and immediately centrifuge at 13,000 rpm for 20 minutes at 2-8°C.

4)

Transfer lysates to a clean tube. Determine the cell lysate concentration using a total protein assay (BCA Protein Assay Kit, Pierce, Prod# 23227). Aliquot the lysates and store at – 80°C.

2. Extracting Protein from Crude Tissue 1)

Transfer approximately 100 mg crude tissue into a tube with 1 ml 1X Cell Lysis Buffer (2X Cell Lysis Buffer should be diluted 2fold with deionized or distilled water).

2)

Homogenize the tissue according to the homogenizer manufacturer’s instructions.

3)

Transfer extracts to microcentrifuge tubes and centrifuge for 20 min at 13,000 rpm (4°C). 8

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

Note: If the supernatant appears to be cloudy, transfer the supernatant 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 -80°C for 20 minutes. Remove from the freezer and immediately centrifuge at 13,000 rpm for 20 minutes at 2-8°C. 4)

Transfer the supernatant to a clean tube and store at – 80°C.

B. 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.

C. Incubations and Washes • • • •

Completely cover membranes with sample or buffer during incubation and cover the Plastic Incubation Tray with the 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 on page 10 (sample incubation) or step 7 on page 11 (HRP-Conjugated Streptavidin incubation) may be done at 4 °C overnight.

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

IV. Protocol

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

9 cm

30 columns x 36 rows

Assay Diagram*

* For serum or plasma, refer to Steps 2, 4, 5 and 6 only. 10 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

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

Prepare dialysis buffer (1X PBS, pH 8.0) by dissolving 0.6 g KCl, 24 g NaCl, 0.6 g KH2PO4 and 3.45 g Na2HPO4 in 2500 ml deionized 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 separate Dialysis Vial (Item A-2). Loading volumes are as follows: 0.1-0.2 ml cell or tissue lysate (~ 1-2 mg/ml total protein) per vial for dialyzing; 30 μl serum or plasma + 120 μl 1X PBS, pH=8 (5-fold dilution) per vial for dialyzing. Carefully place all Dialysis Vials into the Floating Rack.

Note: If the sample appears to be cloudy, transfer the supernatant to a clean tube, centrifuge again at 13,000 rpm for 20 minutes at 2-8°C. 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 stirring the dialysis buffer gently. Then exchange the dialysis buffer with fresh buffer and repeat dialysis for at least 3 hours at 4°C. Transfer the dialyzed samples into clean eppendorf tubes. Centrifuge dialyzed samples for 5 minutes at 10,000 rpm to remove any particulates or precipitates, and then transfer and combine 11

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

each sample into one clean eppendorf tube. Mix well by gently pipetting. Note: The sample volume may change during dialysis. Note: Dialysis procedure may proceed overnight. Note: Determine the total protein concentration for cell or tissue lysates after the dialysis procedure (Step 3). RayBiotech recommends a BCA total protein assay (e.g. Pierce BCA Protein Assay Kit, cat# 23227). 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 by briefly centrifuging the Labeling Reagent vial (Item B) and adding 500 µl 1X PBS (pH 8.0) into the vial. Pipette up and down or vortex briefly to dissolve the lyophilized reagent.

5)

Add 1X Labeling Reagent to dialyzed samples. a. For labeling cell or tissue lysates: transfer 90 µg total protein (e.g., 30 µl of 2 mg/ml) cell or tissue lysates into a tube and add enough 1X PBS for a total volume of 260 µl. Then add 10 µl 1X Labeling Reagent. 12

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

b. For labeling serum or plasma: Add 65 μl of 1X Labeling Reagent Solution into a new tube containing 105 μl dialyzed serum or plasma sample and 80 μl 1X PBS. Note: To normalize serum/plasma concentrations during biotinylation, measure the sample volume before and after dialysis. Then adjust the volumes of the dialyzed serum/plasma and 1X PBS Buffer to compensate. For example, if the sample volume increases 1.4 fold (from 150 μl to 210 μl, see “Dialysis of Sample” on page 11) after dialysis, then use 1.4 fold more serum/plasma (1.4*105 μl = 147 μl) and reduce the added 1X PBS to 38 μl (80 μl - 42 μl = 38 μl). 6)

Incubate the reaction solution at room temperature for 30 minutes with gentle rocking or shaking. Mix the reaction solution by gently tapping the tube every 5 minutes.

7)

Add 5 µl Stop Solution (Item D) into each reaction tube and then use the Spin Column (Item J-2) as follows 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 16 ml conical collection tube for J-2. b) Centrifuge the Spin Column at 1,000 x g for 3 minutes to remove the storage solution. 13

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

Note: The resin should appear compacted after centrifugation. c) Add 1 ml 1X PBS (pH=8.0) into the Item J-2, 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 15 ml conical collection tube and slowly load biotinylated cell/tissue or serum/plasma to the center of the compact resin bed. Note: The maximal sample volume is 700 ul for J-2 each Spin Column. Do not load over maximal sample volume 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 15 ml conical collection tube. Store the sample at -80 0C until you are ready to proceed with the assay. Discard the Spin Column after use. C. Blocking and Incubation 8)

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. 14

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

9)

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.

10) Aspirate the 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. * It is recommended to use 8 ml of 80-fold diluted biotinlabeled cell/tissue lysate or 8 ml 70 fold diluted biotin-labeled serum/plasma. Dilute sample using Blocking Buffer. Note: The optimal concentration of sample used will depend on the abundance of target 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: Incubation may be done at room temperature with gentle shaking for 2 hours or overnight at 4°C. 11) Dilute 20X Wash Buffer 1 (Item G) 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. 12) Aspirate the 1X Wash Buffer 1 from each tray. Dilute 20X Wash Buffer 2 (Item H) with deionized or distilled water to prepare 15 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

the 1X Wash Buffer 2. Wash 3 times with 20 ml of 1X Wash Buffer 2 at room temperature with gentle shaking. 13) Aspirate the 1X Wash Buffer 2 from each tray. 14) Prepare the HRP-Conjugated Streptavidin. Briefly spin down the tube containing the 500X HRP-Conjugated Streptavidin (Item I) immediately before use. Dilute the 500X HRPConjugated Streptavidin with Blocking Buffer to prepare the 1X HRP-Conjugated Streptavidin. Pipette up and down to mix gently. Add 8 ml of 1X HRP-Conjugated 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. 15) Incubate at room temperature with gentle shaking for 2 hours. Note: incubation may be done overnight at 4 oC. 16) Wash as directed in steps 11 through 13. D. Detection Note: Do not let the membrane dry out during detection. The detection process must be completed within 40 minutes without stopping. 16 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

17) 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 on 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 onto 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. 18) 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. 19) 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 the exposure time (e.g., 5–30 seconds). If the signals are too weak, increase the exposure time (e.g., 5–20 min or overnight) or re-incubate membranes overnight with 1X HRPConjugated Streptavidin, and repeat detection on the second day. 17 RayBio® L-Series Human Antibody Array 507 or 493 Protocol

20) 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 by emailing [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

18 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

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 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 

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 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

21 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

22 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

23 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 Arrays. This tool will not only assist in compiling and organizing your data, but also reduces your calculations to a “copy and paste.” Contact RayBiotech, Inc. at 770-729-2992 or [email protected] for ordering information.

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

L-507

L-493

Normal human sera were dialyzed, labeled with biotin and incubation with antibody array according to the protocol in the manual. The images captured using a chemiluminescence imaging system (UVP BioImaging Systems).

25 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.

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. Sample is too dilute.

4. Increase sample concentration

5. Other.

1.Check if there were any contamination with any solution containing amines in biotin-labeling step 2. Slightly increase 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.

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

VIII. Reference List Scheel C, Eaton EN, Li, SH, et al. Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Cell. 2011; 145(6): 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 hepatocellular 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. Zhong J, Krawczyk SA, Chaerkady R, et al. Temporal Profiling of the Secretome during adipogenesis in humans. Journal of Proteome Research. 2010; 9(10): 5228–5238.

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

Chowdury UR, Madden BJ, Charlesworth MC, et al. Proteomic Analysis of Human Aqueous Humor. Invest Ophthalmol Visual Sci. 2010; 51(10): 4921–4931. Wei Y, Cui C, Lainscak M, et al. Type-specific dysregulation of matrix metalloproteinases and their tissue inhibitors in endstage heart failure patients: relationship between MMP-10 and LV remodeling. J Cell Mol Med. 2011; 15(4): 773–782. 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. Temporal Profiling of the Secretome during adipogenesis in humans.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. Hou Z, Sun L, Gao L, et al. Cytokine array analysis of peritoneal fluid between women with endometriosis of different stages and those without endometriosis. Biomarkers. 2009; 14(8): 604-618. Tang YL, Zhu W, Cheng M, et al. Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 expression. Circ Res. 2009; 104(10): 1209-1216.

28 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 the 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 these 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.

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

This product is for research use only.

©2004 RayBiotech, Inc.

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