Antibody MicroArray
Antibody MicroArray User’s Guide Antibody Microarray User’s Guide
Antibody Array Material/Reagent
Quantity
Purpose
2 slides
Microarray
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
Antibody Microarray
Storage Condition 4°C (3 months) -20°C (one year)
Page 2
Antibody MicroArray - User’s Guide
Antibody Array Assay Kit Catalog CatalogNo. No.
Description Description
Quantity Quantity
AR-KAS0-02 KAS02 AR-KAS0-20 KAS20
Antibody Array Assay Kit, 2 Reactions Antibody Array Assay Kit, 2 Reactions Antibody Array Assay Kit, 20 Reactions Antibody Array Assay Kit, 20 Reactions
2 Reactions 2 Reactions 20 Reactions 20 Reactions
Kit Components Quantity
Purpose
Storage Condition
5 x 1mg
Labeling
-20 °C
60 mL
1L
Blocking
4 °C
Coupling Reagent
2 mL
10 mL
Coupling
-20 °C
Detection Buffer
60 mL
1L
Detection
4 °C
DMF
200 uL
1.5 mL
Labeling
4 °C
1.8g & 0.06g
30g & 0.3g
Coupling
4 °C
Extraction Buffer
1.5 ml
20 mL
Protein extraction
4 °C
Labeling Buffer
500 uL
5 mL
Labeling
4 °C
Stop Reagent
100 uL
1 mL
Stop labeling reaction
4 °C
10X Wash Buffer
100 mL
1L
Washing
4 °C
Material/Reagent
2-Rxn Kit
20-Rxn Kit
Biotin Reagent
1 mg
Blocking Reagent
Dry Milk
Antibody MicroArray - User’s Guide
Page 3
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. Microarray scanner compatible with 3in x 1in (76mm x 25mm) slides Milli-Q Grade Water or dd H2O Petri dishes, 9 cm in diameter, for processing one slide Petri dishes, 15 cm in diameter, for processing two slides Shaker Tissue Homogenizing Tubes
Reagent Preparation 2-RXN Kit (KAS02) 1
1X Wash Solution
In a 1 L reagent bottle, add 100 ml of 10X Wash Buffer to 900 ml of dd H2O 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.
20-RXN Kit (KAS20) 1
1X Wash Solution
Make 1:9 (v:v) dilution of 10X Wash Buffer with ddH2O. Example: add 100 ml of 10X Wash Buffer to 900 ml of ddH2O to make 1L of 1X Wash Solution.
2
Blocking Solution
Add 30 g of Dry Milk to 1 Liter of Blocking Reagent. Shake to mix. Use within one week.
3
Coupling Solution
Add 0.3g of Dry Milk to 10 ml of Coupling Reagent. Shake to mix. Use within one week.
*All reagents must be warmed to room temperature before use.
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Antibody MicroArray - User’s Guide
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
Antibody MicroArray - User’s Guide
Page 5
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 Solution 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)
30
50
2.5
47.5
40
60
3
57
50
70
3.5
66.5
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. Rinse the slide(s) extensively with Milli-Q grade water for 30 seconds or more. 5. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure). 6. Place the Layout Slide on a flat surface. Place the Antibody Array Slide over the 5
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Antibody MicroArray - User’s Guide
Layout Slide with the arrays facing up. Be sure the two slides are properly aligned. 7. Clean LifterSlips with DI water followed by 100% ethanol rinse. Dry with a gentle stream of compressed nitrogen or clean air. 8. Locate the white rails on the LifterSlip. This side will face the arrays. 9. 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. 10. Very slowly pipette at least half of the Protein Coupling Mix (See “Section B.c” for the required amount) onto the slide, 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
LifterSlip Slide Solution Do not pipette under white rails. Only pipette under clear glass.
6
Antibody MicroArray - User’s Guide
Page 7
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
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Antibody MicroArray - User’s Guide
11. Remove the Antibody Array Slide from the Layout Slide. Proceed with next slide. 12. 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. 13. Remove the slide(s) from the incubation chamber. 14. 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. 15. 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. 16. Rinse the slide(s) with Milli-Q grade water. 17. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure).
D. Detection
Ref. GMA-ANTIMAMAN-1207-V1
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 40-60 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.
Belgium • Luxembourg Eurogentec Headquarters 3 +32 4 372 74 00 5 +32 4 372 75 00 u [email protected]
Experience true partnership EUROGENTEC s.a. - LIEGE Science Park - 4102 Seraing - BELGIUM RPM Liège - VAT (BE) 0427.348.346 - Tel.: + 32 4 372 76 65 Fax: + 32 4 264 07 88 - [email protected] - www.eurogentec.com
United Kingdom Eurogentec Ltd. 3 +44 (0) 1794 511 411 5 +44 (0) 1794 522 417 u [email protected]
France Eurogentec France s.a.s.u. 3 +33 2 41 73 33 73 5 +33 2 41 73 10 26 u [email protected]
Germany • Austria Eurogentec Deutschland GmbH 3 +49 221 258 94 55 5 +49 221 258 94 54 u [email protected]
The Netherlands Eurogentec Nederland b.v. 3 +31 43 352 06 98 5 +31 43 354 19 65 u [email protected]
Switzerland Eurogentec s.a. succursale de Genève 3 FR: +32 4 372 74 00 DE: +49 221 258 94 55 5 FR: +32 4 372 75 00 DE: +49 221 258 94 54
Eurogentec North America Inc. 3347 Industrial Court - Suite A San Diego, CA 92121, USA 3 877 387 6436 5 858 793 2666 u [email protected]
8
Antibody Array Material/Reagent
Quantity
Purpose
2 slides
Microarray
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
Antibody Microarray
Storage Condition 4°C (3 months) -20°C (one year)
Page 2
Antibody MicroArray - User’s Guide
Antibody Array Assay Kit Catalog CatalogNo. No.
Description Description
Quantity Quantity
AR-KAS0-02 KAS02 AR-KAS0-20 KAS20
Antibody Array Assay Kit, 2 Reactions Antibody Array Assay Kit, 2 Reactions Antibody Array Assay Kit, 20 Reactions Antibody Array Assay Kit, 20 Reactions
2 Reactions 2 Reactions 20 Reactions 20 Reactions
Kit Components Quantity
Purpose
Storage Condition
5 x 1mg
Labeling
-20 °C
60 mL
1L
Blocking
4 °C
Coupling Reagent
2 mL
10 mL
Coupling
-20 °C
Detection Buffer
60 mL
1L
Detection
4 °C
DMF
200 uL
1.5 mL
Labeling
4 °C
1.8g & 0.06g
30g & 0.3g
Coupling
4 °C
Extraction Buffer
1.5 ml
20 mL
Protein extraction
4 °C
Labeling Buffer
500 uL
5 mL
Labeling
4 °C
Stop Reagent
100 uL
1 mL
Stop labeling reaction
4 °C
10X Wash Buffer
100 mL
1L
Washing
4 °C
Material/Reagent
2-Rxn Kit
20-Rxn Kit
Biotin Reagent
1 mg
Blocking Reagent
Dry Milk
Antibody MicroArray - User’s Guide
Page 3
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. Microarray scanner compatible with 3in x 1in (76mm x 25mm) slides Milli-Q Grade Water or dd H2O Petri dishes, 9 cm in diameter, for processing one slide Petri dishes, 15 cm in diameter, for processing two slides Shaker Tissue Homogenizing Tubes
Reagent Preparation 2-RXN Kit (KAS02) 1
1X Wash Solution
In a 1 L reagent bottle, add 100 ml of 10X Wash Buffer to 900 ml of dd H2O 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.
20-RXN Kit (KAS20) 1
1X Wash Solution
Make 1:9 (v:v) dilution of 10X Wash Buffer with ddH2O. Example: add 100 ml of 10X Wash Buffer to 900 ml of ddH2O to make 1L of 1X Wash Solution.
2
Blocking Solution
Add 30 g of Dry Milk to 1 Liter of Blocking Reagent. Shake to mix. Use within one week.
3
Coupling Solution
Add 0.3g of Dry Milk to 10 ml of Coupling Reagent. Shake to mix. Use within one week.
*All reagents must be warmed to room temperature before use.
Page 4
Antibody MicroArray - User’s Guide
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
Antibody MicroArray - User’s Guide
Page 5
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 Solution 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)
30
50
2.5
47.5
40
60
3
57
50
70
3.5
66.5
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. Rinse the slide(s) extensively with Milli-Q grade water for 30 seconds or more. 5. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure). 6. Place the Layout Slide on a flat surface. Place the Antibody Array Slide over the 5
Page 6
Antibody MicroArray - User’s Guide
Layout Slide with the arrays facing up. Be sure the two slides are properly aligned. 7. Clean LifterSlips with DI water followed by 100% ethanol rinse. Dry with a gentle stream of compressed nitrogen or clean air. 8. Locate the white rails on the LifterSlip. This side will face the arrays. 9. 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. 10. Very slowly pipette at least half of the Protein Coupling Mix (See “Section B.c” for the required amount) onto the slide, 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
LifterSlip Slide Solution Do not pipette under white rails. Only pipette under clear glass.
6
Antibody MicroArray - User’s Guide
Page 7
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
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Antibody MicroArray - User’s Guide
11. Remove the Antibody Array Slide from the Layout Slide. Proceed with next slide. 12. 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. 13. Remove the slide(s) from the incubation chamber. 14. 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. 15. 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. 16. Rinse the slide(s) with Milli-Q grade water. 17. Dry the slide(s) by centrifugation or with compressed nitrogen (less than 20 psi of pressure).
D. Detection
Ref. GMA-ANTIMAMAN-1207-V1
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 40-60 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.
Belgium • Luxembourg Eurogentec Headquarters 3 +32 4 372 74 00 5 +32 4 372 75 00 u [email protected]
Experience true partnership EUROGENTEC s.a. - LIEGE Science Park - 4102 Seraing - BELGIUM RPM Liège - VAT (BE) 0427.348.346 - Tel.: + 32 4 372 76 65 Fax: + 32 4 264 07 88 - [email protected] - www.eurogentec.com
United Kingdom Eurogentec Ltd. 3 +44 (0) 1794 511 411 5 +44 (0) 1794 522 417 u [email protected]
France Eurogentec France s.a.s.u. 3 +33 2 41 73 33 73 5 +33 2 41 73 10 26 u [email protected]
Germany • Austria Eurogentec Deutschland GmbH 3 +49 221 258 94 55 5 +49 221 258 94 54 u [email protected]
The Netherlands Eurogentec Nederland b.v. 3 +31 43 352 06 98 5 +31 43 354 19 65 u [email protected]
Switzerland Eurogentec s.a. succursale de Genève 3 FR: +32 4 372 74 00 DE: +49 221 258 94 55 5 FR: +32 4 372 75 00 DE: +49 221 258 94 54
Eurogentec North America Inc. 3347 Industrial Court - Suite A San Diego, CA 92121, USA 3 877 387 6436 5 858 793 2666 u [email protected]
8
