Protocol. Epoxy Slide For Protein Array Applications. Version 2.1. February 2014

Transcription

1 Protocol Epoxy Slide For Protein Array Applications Version 2.1 February 2014 For Technical Assistance, Please contact Z Biotech, LLC Fitzsimons Bioscience Park Center E. Montview Blvd., Suite 214 Aurora, CO info@zbiotech.com Website:

2 Introduction: Z Biotech provides a full spectrum of high quality microarray slides for printing various microarrays, including protein microarrays, glycan microarrays, reverse-phase protein microarrays and small molecule microarrays. The epoxy-functionalized slides are manufactured with a state-of-the-art CVD process in a class 100 cleanroom on microscope slides that feature an ultra-low fluorescent background. The epoxy slides are highly recommended for proteomics, high-throughput drug screening, and all other applications requiring a premium-high quality microarray surface. For each type of the surface coating, we offer two versions of microarray slides: surface functionalized planar glass slides and 3D slides featured with patterned microwells or micropillars. All of the microarray slides are compatible with contact printing and non-contact microarray spotting tools. For customers to obtain the best microarray results, we also provide validated printing, blocking, and binding buffers for each type of microarray slide. Storage and Handling: Store at room temperature in a sealed pouch prior to use! After opening, seal any unused slides in the reusable pouch with desiccant inside. Avoid contact with the surface of the slides to minimize contamination and abrasion of the surface. Wear gloves and hold slide along the edges. Buffers Required: Printing Buffer: NEPPB, NHS/Epoxy Protein Printing Buffer, 2X concentration Blocking Buffer: 1% BSA in PBST (PBS with 0.05% (v/v) Tween 20, ph 7.4) Assay Buffer: PAAB, Protein Array Assay Buffer,, 2X concentration Wash Buffer: 20 mm Tris-HCl, ph 7.6, 150 mm NaCl, 0.05% Tween 20 Array Printing and Incubation: 1. Print proteins at an appropriate concentration in 1X NEPPB Printing Buffer. A protein probe concentration ranging from 0.1 to 1 mg/ml is recommended to ensure sufficient protein loading and to enable reliable and consistent assay results; 2. Incubate printed slides in a humidified chamber (60% humidity) for 1 hour at room temperature. 3. Printed slide can be stored at -20 C after printing and immobilization, but before washing and blocking. Blocking: 1. Affix a 16-chamber assay adaptor onto slide surface; 2. Block the slide for 1 hour in Blocking Buffer at room temperature with static condition.

3 Binding Assay or Immunoassay: 1. Prepare target samples (i.e., binding proteins or primary antibodies) in an appropriate dilution factor in PAAB Assay Buffer; 2. Remove the Blocking Buffer liquid out of each assay chamber by vacuum suction or pipette; 3. Carefully apply 70 l sample (prepared in step 1) to the appropriate chamber; 4. Cover the chambers with an adhesive seal to prevent evaporation during incubation; 5. Incubate slide on a rotating shaker at 60 rpm for 1 hour; 6. After 1 hour of incubation, remove slide from shaker and remove cover; 7. Remove sample from each chamber by vacuum suction or pipette; 8. Wash each chamber by adding 100 l Wash Buffer twice (5-minutes incubation for each wash); For biotinylated samples, following procedures below to stain the slides: 9. Add 70 l of Cy3 or Cy5 labeled Streptavidin (or Avidin) at 0.5 g/ml concentration to each assay chamber; 10. Cover the chambers with an adhesive seal; 11. Incubate the slide on the rotating shaker at 60 rpm for 30 minutes; 12. Remove the sample from each chamber by vacuum suction or pipette; 13. Wash each chamber by adding 100 l Wash Buffer; 14. Remove the 16-chamber adaptor and wash the slides twice in a Coplin jar of 100 ml Wash Buffer (5- minutes incubation for each wash); 15. Rinse the slide with 100 ml 0.01x PBS and DI water respectively; 16. Spin the slide in slide centrifuge for 15 seconds; 17. Scan the slide with a fluorescent microarray scanner at the appropriate wavelengths and setting; 18. Save the scanning image and process the data with appropriate quantitation software. For unlabeled antibody samples, following procedures below to stain the slides: 10. Add 70 l of Cy3 or Cy5 labeled secondary antibody at 0.5 g/ml concentration to each assay chamber; 11. Cover the chambers with an adhesive seal; 12. Incubate the slide on the rotating shaker at 60 rpm for 30 minutes; 13. Remove the sample from each chamber by vacuum suction or pipette; 14. Wash each chamber by adding 100 l Wash Buffer; 15. Remove the 16-chamber adaptor and wash the slides twice in a Coplin jar of 100 ml Wash Buffer (5- minutes incubation for each wash); 16. Rinse the slide with 100 ml 0.01x PBS and DI water respectively; 17. Spin the slide in slide centrifuge for 15 seconds;

4 18. Scan the slide with a fluorescent microarray scanner at the appropriate wavelengths and setting; 19. Save the scanning image and process the data with appropriate quantitation software.

5 Protocol Epoxysilane Slide For Glycan Array Application Version 2.1 March 2014 For Technical Assistance, Please contact Z Biotech, LLC Fitzsimons Bioscience Park Center E. Montview Blvd., Suite 214 Aurora, CO info@zbiotech.com Website:

6 Introduction: Z Biotech provides a full spectrum of high quality microarray slides for printing various microarrays, including protein microarrays, glycan microarrays, reverse-phase protein microarrays and small molecule microarrays. The epoxysilane slides are manufactured with a state-of-the-art CVD process in a class 100 cleanroom on microscope slides that feature an ultra-low fluorescent background. The epoxysilane slides offer high loading capacity and extremely low background for the immobilization of proteins (antibodies and antigens), amine-modified glycans and amine-modified small chemical molecules. For each type of the surface coating, we offer two versions of microarray slides: surface functionalized planar glass slides and 3-D slides featured with patterned microwells or micropillars. All of the microarray slides are compatible with contact printing and non-contact microarray spotting tools. For customers to obtain the best microarray results, we also provide validated printing, blocking, and assay buffers for each type of microarray slide. Storage and Handling: Store at room temperature in a sealed pouch prior to use! After opening, seal any unused slides in the reusable pouch with desiccant inside. Avoid contact with the surface of the slides to minimize contamination and abrasion of the surface. Wear gloves and hold slide along the edges. Buffers Required: Printing Buffer: NGPB, NHS Glycan Printing Buffer, 2X concentration Blocking Buffer: Planar and Micropillar Slides: NGBB, NHS Glycan Blocking Buffer, 1X concentration (or 1% BSA in PBST (PBS with 0.05% (v/v) Tween-20, ph 7.4)) 3-D Micowell Slides: MNGBB, Microwell NHS Glycan Blocking Buffer, 1X concentration (or 1% Casein in PBST (PBS with 0.05% (v/v) Tween-20, ph 7.4)) Assay Buffer: NHGAB, NHS/Hydrazide Glycan Assay Buffer, 1X concentration Wash Buffer: 20 mm Tris-HCl, ph 7.6, 150 mm NaCl, 0.05% Tween 20 (TBST) Array Printing and Glycan Immobilization: 1. Prepare glycans containing amino group at an appropriate concentration in Printing Buffer. A glycan probe concentration ranging from 0.01 to 1 mm is recommended to ensure sufficient glycan loading and to enable reliable and consistent assay results; The epoxysilane slides provide covalent attachment of amine-modified glycans. The coupling efficiency of the covalent chemistry depends on a number of factors, including ph, glycan print concentration, and the nature of the glycan itself. The epoxysilane slides are compatible with all microarray printing or spotting methods, including contact printing and piezo or ink-jet technologies.

7 2. Print the glycan probe solution on the epoxysilane slides in humidified chamber (60% humidity). 3. Incubate printed slides in a humidified chamber (50% humidity) for 1 hour at 50 C; or leave the printed slides stayed in a humidified chamber (50% humidity) at room temperature for overnight. Blocking: 1. Wash the printed slide with DI water for 2 minutes; 2. Block the slide for 1 hour in Blocking Buffer at room temperature with gentle shaking at 60 rpm; 3. Rinse the slide in DI water for 2 minutes; 4. Spin the slide in a slide centrifuge for 15 seconds. Binding Assay or Immunoassay: 1. Affix a 16-chamber assay adaptor onto slide surface; 2. Hydrate slide with 100 l Wash Buffer for 5 minutes at room temperature with shaking at 60 rpm; 3. Remove the Wash Buffer by vacuum suction or pipetting the liquid out of each assay chamber; 4. Prepare samples (biotinylated or fluorescence-labeled) in NHGAB Assay Buffer; 5. Apply 70 l samples (prepared in step 4) to the appropriate chamber; 6. Cover the chamber windows with an adhesive seal to prevent evaporation during incubation; 7. Incubate the slide on the rotating shaker at 60 rpm for 1 hour; 8. After 1 hour incubation, remove the slide from shaker and remove cover; 9. Remove sample from each chamber by vacuum suction or pipetting out; 10. Wash each chamber by adding 100 l Wash Buffer twice (5-minutes incubation for each wash); For Biotinylated samples, following procedures below to stain the slides: 11. Add 70 l of Cy3 or Cy5 labeled streptavidin at 0.5 g/ml concentration to each assay chamber; 12. Cover the chambers with an adhesive seal; 13. Incubate the slide on the rotating shaker at 60 rpm for 30 minutes; 14. Remove the sample from each chamber by vacuum suction or pipetting out; 15. Wash each chamber by adding 100 l Wash Buffer; 16. Remove the 16-chamber adaptor and wash the slide twice in a Coplin jar of 100 ml Wash Buffer (5- minutes incubation for each wash); 17. Rinse the slide with 100 ml 0.01x PBS and DI water respectively; 18. Spin the slide in a slide centrifuge for 15 seconds; 19. Scan the slide with a fluorescent microarray scanner at the appropriate wavelengths and setting; 20. Save the scanning image and process the data with appropriate quantitation software. For unlabeled antibody samples, following procedures below to stain the slides:

8 11. Add 70 l of Cy3 or Cy5 labeled secondary antibody at 0.5 g/ml concentration to each assay chamber; 12. Cover the chambers with an adhesive seal; 13. Incubate the slide on the rotating shaker at 60 rpm for 30 minutes; 14. Remove the sample from each chamber by vacuum suction or pipette; 15. Wash each chamber by adding 100 l Wash Buffer; 16. Remove the 16-chamber adaptor and wash the slides twice in a Coplin jar of 100 ml Wash Buffer (5- minutes incubation for each wash); 17. Rinse the slide with 100 ml 0.01x PBS and DI water respectively; 18. Spin the slide in slide centrifuge for 15 seconds; 19. Scan the slide with a fluorescent microarray scanner at the appropriate wavelengths and setting; 20. Save the scanning image and process the data with appropriate quantitation software.