Antibody List. Array Name: Tyrosine Kinase Adaptor Phosphorylation Antibody Array Cat. #: PTK098 Total Antibodies: 98 Revision: A1

Transcription

1 Array Name: Tyrosine Kinase Adaptor Phosphorylation Antibody Array Cat. #: PTK098 Total Antibodies: 98 Revision: A1 Antibody List ID Antibody Name A Beta actin E Empty G GAPDH N Negative control P Positive control 1 MEK-2(Phosph-Phospho-Thr394) 2 FAK(Phospho-Tyr861) 3 Chk2(Phospho-Thr68) 4 HER2(Phospho-Tyr877) 5 HER2(Phospho-Tyr1221/Phospho-Tyr1222) 6 HER2(Phospho-Tyr1248) 7 EGFR(Phospho-Ser1070) 8 EGFR(Phospho-Tyr1092) 9 VEGFR2(Phospho-Tyr1175) 10 VEGFR2(Phospho-Tyr1214) 11 VEGFR2(Phospho-Tyr951) 12 IGF-1R (Phospho-Tyr1161) 13 IGF-1R (Phospho-Tyr1165/Phospho-Tyr1166) 14 Src(Phospho-Tyr418) 15 Chk1(Phospho-Ser345) 16 FAK(Phospho-Tyr925) 17 Chk1(Phospho-Ser280) 18 Chk1(Phospho-Ser317) 19 Chk2(Phospho-Ser516) 20 TYK2(Phospho-Tyr1054) 21 JAK1(Phospho-Tyr1022) 22 JAK2(Phospho-Tyr221) 23 JAK2(Phospho-Tyr1007) 24 Src(Phospho-Tyr529) 25 Zap-70(Phospho-Tyr319) 26 Zap-70(Phospho-Tyr493) 27 MEK1(Phospho-Ser221) 28 MEK1(Phospho-Ser217) 29 Pyk2(Phospho-Tyr402) 30 EGFR(Phospho-Tyr1172) 31 Stathmin 1(Phospho-Ser24) 32 Stathmin 1(Phospho-Ser37) 33 Met(Phospho-Tyr1234) 34 EGFR(Phospho-Tyr197) 35 EGFR(Phospho-Tyr869) 36 Stathmin 1(Phospho-Ser15) 37 Met(Phospho-Tyr1349) 38 c-kit(phospho-tyr721) 39 SAPK/JNK(Phospho-Thr183) 40 SAPK/JNK(Phospho-Tyr185) 41 P38 MAPK(Phospho-Thr180) 42 P38 MAPK (Phospho-Tyr182) 43 EGFR(Phospho-Tyr1110) 44 PKR(Phospho-Thr446) 45 CaMKII (Phospho-Thr286) 46 PKR(Phospho-Thr451) 47 MEK1(Phospho-Thr291)

2 Array Layout c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 r1 P E E P r2 P E E P Block 1 r3 P E E P r4 P E E P r5 P E E P r6 P E E P Block 1 Block 2 Block 3 Block 4 Block 5 c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 r1 p E E P r2 p E E P Block 2 r3 p E E P r4 p E E P r5 p E E P r6 p E E P c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 r1 P E E P r2 P E E P Block 3 r3 P E E P r4 P E E P r5 P E E P r6 P E E P Barcode Label c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 r1 P E E P r2 P E E P Block 4 r3 P E E P r4 P E E P r5 P E E P r6 P E E P c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 c16 c17 c18 c19 c20 c21 r1 P E E N N A G P r2 P E E N N A G P Block 5 r3 P E E N N A G P r4 P E E N N A G P r5 P E E N N A G P r6 P E E N N A G P

3 Antibody Microarray User s Guide (3.10) Antibody Array Material/Reagent Quantity Purpose Storage Condition Antibody Microarray 2 slides Microarray 4 C (3 months) -20 C (one year) Layout Slide 1 Array layout Room temperature LifterSlips 2 Coupling Room temperature Gal File (CD) 1 Data Analysis Room temperature User s Guide (CD) 1 Instructions Room temperature 1

4 Antibody Array Assay Kit Catalog No. Description Quantity KAS02 Antibody Array Assay Kit, 2 Reactions 2 Reactions Kit Components Material/Reagent Quantity Purpose Storage Condition Biotin Reagent 1 mg Labeling -20 C Blocking Reagent 60 ml Blocking 4 C Coupling Reagent 2 ml Coupling -20 C Detection Buffer 60 ml Detection 4 C DMF 200 ul Labeling 4 C Extraction Buffer 1.5 ml Protein extraction 4 C Dry Milk 1.8g & 0.06g Coupling 4 C Labeling Buffer 500 ul Labeling 4 C Stop Reagent 100 ul Stop labeling reaction 4 C 10X Wash Buffer 100 ml Washing 4 C 2

5 Additional Material Required 1X PBS (ph=7.4) BCA Protein Assay Kit (Pierce, Cat. #: 23227) Cy3-Streptavidin (GE Healthcare, Cat. #: PA43001) Centrifuge Compressed nitrogen/clean air or Centrifuge with swinging buckets Humidity Chamber with 100% relative humidity A simple humidity chamber can be easily put together with a container with tight seals and water. For example, a pipette tip box can be easily converted to a humidity chamber. Remove the tip rack from the box. Add water to cover the bottom of the container. Replace the tip rack and close the lid. For humidity treatment, place two slides on the tip rack and close the lid tightly. Milli-Q Grade Water or dd H 2 O Petri dishes, 9 cm in diameter, for processing one slide Petri dishes, 15 cm in diameter, for processing two slides Reagent bottle, plastic or glass, 1 L Reagent tube, plastic or glass, 5 ml Two reagent bottles, plastic or glass, 60 ml Shaker Tissue Homogenizing Tubes Reagent Preparation 1 1X Wash Solution In a 1 L reagent bottle, add 100 ml of 10X Wash Buffer to 900 ml of dd H 2 O to make 1L of 1X Wash Solution. 2 Blocking Solution Add 1.8 g of Dry Milk to 60 ml of Blocking Reagent. Shake to mix. Use within one week. 3 Coupling Solution Add 0.06g of Dry Milk to 2 ml of Coupling Reagent. Shake to mix. Use within one week. *All reagents must be warmed to room temperature before use. 3

6 Protocol Detection by Cy3-Streptavidin *All reagents must be warmed to room temperature before use. A. Protein Extraction a. Extraction from Body Fluid 1. Centrifuge the sample at 10,000 x g for 10 minutes at 4 C. 2. Transfer the supernatant to a clean tube. 3. Measure the protein concentration using the BCA Protein Assay Kit (Pierce, Cat #: 23227). 4. Proceed immediately to Step B (Protein Labeling) or store the samples at -80 C. b. Extraction from Cells 1. Harvest the cells by centrifugation. 2. Wash the cells with 1X PBS and centrifuge at 4 C. Aspirate and discard the supernatant. 3. Wash the cells two more times with cold 1X PBS and centrifuge at 4 C. 4. Keep the cell pellet on ice for 5 minutes. 5. Add Extraction Buffer to the cell pellet and mix thoroughly by pipetting. 6. Incubate the mixture on ice for 10 minutes with occasional mixing. 7. Centrifuge the mixture at 10,000 x g for 30 minutes at 4 C. 8. Transfer the supernatant to a clean tube. 9. Measure the protein concentration using BCA Protein Assay Kit. 10. Proceed immediately to Step B (Protein Labeling) or store the samples at -80 C. c. Extraction from Tissues 1. Place frozen tissues in a homogenizing tube. 2. Add a sufficient amount of Extraction Buffer to cover the tissue. 3. Homogenize the tissue on ice. 4. Transfer the homogenate to a centrifuge tube. 5. Centrifuge the homogenate at 10,000 x g for 30 minutes at 4 C. 6. Transfer the supernatant to a clean tube. 7. Measure the protein concentration using BCA Protein Assay Kit. 8. Proceed immediately to Step B (Protein Labeling) or store the samples at -80 C. B. Protein Labeling a. Biotinylation of Protein Samples 1. Briefly centrifuge Biotin Reagent before use. 2. Add 100 ul of DMF (N,N-Dimethylformamide) to 1 mg of Biotin Reagent to give a concentration of 10 ug/ul. Label this solution as Biotin/DMF. 4

7 3. Aliquot 10 ul (or less) of protein sample that contains 100 ug (or less) of protein. 4. Add 40 ul Labeling Buffer to the protein sample to bring the volume to 50 ul. 5. Add 1.43 ul of the Biotin/DMF solution to the protein sample. Note: The ratio of Biotin Reagent to the protein sample should be 1:7 (w:w). Determine the amount of Biotin Reagent needed if a different amount of protein is used. 6. Mix and incubate at room temperature for two hours with mixing or shaking. 7. Add 25 ul of Stop Reagent. Incubate for 30 minutes at room temperature with mixing or shaking. b. Measuring Biotin Labeled Proteins Measure the concentration of biotin labeled proteins with BAC Protein Assay Reagent Kit. Important: Stop Reagent should be used as Blank when measuring. c. Preparing Protein Coupling Mix Note: We recommend the Biotin labeled proteins be diluted in 1:20 ratio with Coupling Solution (See Reagent Preparation ) before it can be applied to the arrays for conjugation. The dilution factor may vary with your samples. Optimization with different dilutions (1:5, 1:10, 1:100, etc.) is recommended to determine the optimal dilution factor. The amount of Protein Coupling Mix needed varies with the size of LifterSlips. 1. Use the chart below to determine the appropriate amount of Protein Coupling Mix needed for each slide. 2. Prepare the Mix by adding the desired amount of biotin labeled protein to Coupling Solution. If you are working with multiple slides, prepare more accordingly. 3. Store the remaining biotin labeled protein at -80 C for future use. LifterSlip Size Protein Coupling Mix Needed (ul) Biotin Labeled Protein (ul) Coupling Solution (ul) C. Conjugation of Biotin Labeled Protein to Antibody Microarray 1. Remove Antibody Microarrays from the freezer. Important: Allow the slides to warm up to room temperature (30 minutes to 1 hour) before opening the package. 2. Blocking: Use a 9-cm Petri dish for a single slide and 15-cm Petri dish for two slides. Add 30 ml of Blocking Solution (See Reagent Preparation ) into a 9-cm Petri dish or 60 ml to a 15-cm Petri dish. Submerge the slide(s) in the Blocking Solution. Incubate on an orbital shaker set to rotate at 55 rpm for 30 minutes at room temperature. 3. Discard the Blocking Solution. 4. Wash the slide(s) by adding 30 ml (single slide) or 60 ml (two slides) of 1X Wash Solution (See Reagent Preparation ) to the slide(s) in Petri dish. Incubate on an orbital shaker set to rotate at 55 rpm for 5 minutes at room temperature. 5

8 5. Rinse the slide(s) with Milli-Q grade water. 6. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure). 7. Place the Layout Slide on a flat surface. Place the Antibody Array Slide over the Layout Slide with the arrays facing up. Be sure the two slides are properly aligned. 8. Clean LifterSlips with DI water followed by 100% ethanol rinse. Dry with a gentle stream of compressed nitrogen or clean air. 9. Locate the white rails on the LifterSlip. This side will face the arrays. 10. Place a cleaned LifterSlip over the arrayed area with white rails facing down. Be sure to align LifterSlip s two sides with white rails with the long sides of the slide. 11. Very slowly pipette at least half of the Protein Coupling Mix onto the slide (See Section B.c for the required amount), just adjacent to one corner of the LifterSlip. Pipette the remaining solution under the opposite corners. Capillary action will allow the solution to wick under the LifterSlip, yielding uniform coverage of the microarray. Pipetting slowing and gently often helps filling and avoid trapped air bubbles. Tip Tip Solution LifterSlip Slide Do not pipette under white rails. Only pipette under clear glass. 6

9 Solution thinly covers the array Begin a very slow injection of solution from one corner of the LifterSlip the pipette tip should be placed as close as possible to the LifterSlip/Slide junction but not touching. As the solution is expelled from the pipette tip, it will spread under the LifterSlip via capillary action. The array is now covered with a thin layer of solution and the remaining solution can be evenly dispensed at the other corners of the slip: 1) move pipette tip to the diagonal corner of the slip and pipette about one half of the remaining solution. 2) repeat this at the opposite corner. 7

10 12. Remove the Antibody Array Slide from the Layout Slide. Proceed with next slide. 13. Incubate the array(s) at room temperature in a humidity chamber with 100% relative humidity for 2 hours or at 4 C overnight. Incubation time may be extended if desired. 14. Remove the slide(s) from the incubation chamber. 15. Add 50 ml of 1X Wash Solution in a container that allows you to dip the slide(s) up and down. Remove the LifterSlip(s) by dipping the slide(s) the solution. The LifterSlip(s) should slide off automatically. 16. Wash slides by adding 30 ml of 1X Wash Solution (single slide) to a 9-cm Petri dish or 60 ml of 1X Wash Solution (two slides) to a 15-cm Petri dish. Submerge the slide(s) into the 1X Wash Solution. Incubate on an orbital shaker set to rotate at 55 rpm for 10 minutes at room temperature. Discard the wash solution. Repeat the wash step twice. 17. Rinse the slide(s) with Milli-Q grade water. 18. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure). D. Detection 1. Add 60 ul of Cy3-Streptavidin (0.5 mg/ml) to the 60-ml bottle containing Detection Buffer. 2. Load 30 ml of Cy3-Streptavidn Solution into a 9-cm Petri dish for a single slide or 60 ml into a 15-cm Petri dish for two slides. 3. Submerge the slide(s) in the Cy3-Streptavidin solution. Incubate on an orbital shaker set to rotate at 55 rpm for minutes at room temperature in the dark or covered with aluminum foil. 4. Wash the slide(s) by adding 30 ml of 1X Wash Solution (single slide) to a 9-cm Petri dish or 60 ml of 1X Wash Solution (two slides) to a 15-cm Petri dish. Submerge the slide(s) in the Wash Solution. Incubate on an orbital shaker set at 55 rpm for 10 minutes at room temperature. Discard the wash solution. Repeat the wash step twice. 5. Rinse the slide(s) with Milli-Q grade water. 6. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure). 7. The slide(s) is now ready for scanning. 8

11 48 PKCδ (Phospho-Ser645) 49 PKCθ(Phospho-Ser676) 50 MEK-2(Ab-394) 51 HER2(Ab-877) 52 HER2(Ab-1221/1222) 53 HER2(Ab-1248) 54 EGFR(Ab-1070) 55 EGFR(Ab-1092) 56 FAK(Ab-861) 57 VEGFR2(Ab-1175) 58 VEGFR2(Ab-1214) 59 VEGFR2(Ab-951) 60 IGF-1R (Ab-1161) 61 IGF-1R (Ab-1165/1166) 62 Chk2(Ab-68) 63 Chk1(Ab-280) 64 Chk1(Ab-317) 65 Src(Ab-418) 66 Chk2(Ab-516) 67 TYK2(Ab-1054) 68 JAK1(Ab-1022) 69 JAK2(Ab-221) 70 JAK2(Ab-1007) 71 Chk1(Ab-345) 72 FAK(Ab-925) 73 Src(Ab-529) 74 Zap-70(Ab-319) 75 Zap-70(Ab-493) 76 MEK1(Ab-221) 77 MEK1(Ab-217) 78 FAK(Ab-397) 79 Pyk2(Ab-402) 80 EGFR(Ab-1172) 81 Stathmin 1(Ab-24) 82 Stathmin 1(Ab-37) 83 Met(Ab-1234) 84 EGFR(Ab-197) 85 EGFR(Ab-869) 86 Stathmin 1(Ab-15) 87 Met(Ab-1349) 88 c-kit(ab-721) 89 SAPK/JNK(Ab-183) 90 SAPK/JNK(Ab-185) 91 P38 MAPK(Ab-182) 92 EGFR(Ab-1110) 93 PKR(Ab-446) 94 CaMKII (Ab-286) 95 PKR(Ab-451) 96 MEK1(Ab-291) 97 PKCδ (Ab-645) 98 PKCθ(Ab-676)