Protocol for Minichip hybridization. Equipment and Reagents needed: BLOCKING SOLUTION (2% [w/v]) CY-3 STREPTAVIDIN

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1 Protocol for Minichip hybridization Equipment and Reagents needed: 1,000mL or 250mL Filter Units (PES Membrane) (VWR, ) 1,000mL or 250mL Receiver Units (VWR, / ) Weighing Plate (VWR, ) 0.2mL Polypropylene PCR Tube Strips (Corning, PCR-0208-CP-C) Falcon 96-Well Library Storage Plates, Polypropylene (Corning, ) 200uL Pipette Tips (VWR, ) 1,000uL Pipette Tips (VWR, ) 10mL Serological Pipettes (VWR, ) 25mL Serological Pipettes (VWR, ) Nunc Rectangular 4-Well Dish Plates (Thermo Scientific, ) Microscope Slide Boxes, Blue (VWR, ) Reagent Reservoirs, Polystyrene, Sterile (Corning, ) Reynolds Wrap Aluminum Foil (VWR, ) 50mL Centrifuge Tubes (VWR, ) ProPlate 16 Well Slide Module 7mm X 7mm, Delrin Snap Clips (Grace Bio-Labs, ) Pyrex Media Bottles, Graduated, Corning (Corning, L) Fisherbrand Microscope Slide Box (Fisher, ) 1.5mL Black Microcentrifuge Tubes DNase RNase Free (Argos Technologies, T7100BK) 1.5mL Eppendorf Flex-Tubes Disposable Microcentrifuge Tubes, Graduated (VWR, ) Cy3 Streptavidin (Jackson ImmunoResearch Laboratories, ) Alexa Fluor 555 Goat anti-rabbit IgG (H+L) (Invitrogen, A-21428) Alexa Fluor 647-conjugated AffiniPure F(ab )2 Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, ) His-tag Antibody, pab, Rabbit (GenScript, A ) Anti-GST, S/japonicum Form Rabbit Polyclonal (Millipore, ab3282) IgG-Free, Protease-Free Bovine Serum Albumin (Jackson ImmunoResearch Laboratories, ) Tween 20 (Sigma-Aldrich, P ML) TBS, 10X (Tris Buffered Saline) (Corning, CM) BLOCKING SOLUTION (2% [w/v]) 1. Determine the amount of blocking solution to be prepared and calculate amount of IgG-Free, Protease-Free Bovine Serum Albumin (BSA) needed to make a 2% [w/v] solution. 2. Dissolve the BSA in TBS-T buffer (0.05% Tween 20). 3. Vacuum filter to sterilize. 4. Store the filtered solution at 4 C. CY-3 STREPTAVIDIN ***Photosensitive Reagent, protect from light *** 1. Dissolve 1 mg/ml Cy-3 SA in ddh2o. 2. Store in 1.5 ml Black microcentrifuge tube at 4 C.

2 ALEXA FLUOR 555 GOAT ANTI-RABBIT IgG (H+L) ***Photosensitive Reagent, protect from light *** 1. Dilute Alexa Fluor 555 Goat anti-rabbit IgG (H+L) to 1 mg/ml in ddh2o. 2. Place in black microcentrifuge tube and store at 4 C. ALEXA FLUOR 647-CONJUGATED AFFINIPURE F(AB )2 FRAGMENT GOAT ANTI-MOUSE IgG (H+L) ***Photosensitive Reagent, protect from light*** 1. Dilute Alexa Fluor 647-conjugated AffiniPure F(AB ) Fragment Goat anti-mouse IgG (H+L) to 1 mg/ml in ddh2o. 2. Place in black microcentrifuge tube and store at 4 C. HIS-TAG ANTIBODY, pab, RABBIT 1. Dilute His-tag Antibody, pab, Rabbit to 1 mg/ml in ddh2o. 2. Prepare a 1:10 dilution of the 1mg/mL stock in ddh2o. 3. Store the solutions at 4 C. ANTI-GST, S/JAPONICUM FORM RABBIT POLYCLONAL 1. Dilute Anti-GST, S/japonicum form rabbit polyclonal to 1 mg/ml in ddh2o. 2. Prepare a 1:10 dilution of the 1mg/mL stock in ddh2o. 3. Store the solutions at 4 C. TRIS BUFFERED SALINE Tween 20 (TBS-T), 2 L 1. Combine 1,800mL ddh2o, 200mL 10X TBS and 5mL 20% Tween 20. HYBRIDIZATION PROCEDURE ***Avoid condensation or dryness at all times*** SLIDE PREPARATION Select the MiniChip Batch to be used in advance Take out the exact amount of Nunc Rectangular 4-Well Dish Plates to be used for the MiniChip Run. Label accordingly (Permanent Marker)

3 7.1.3 Place Blocking Solution out of Refrigerator Storage Maintain slides in -80 C or dry ice until Blocking Stage Take slide from mailer Note: DO NOT under any circumstance touch the array surface with gloves. If this is to occur protein printed on array will be smudged and unusable. Always handle arrays on the side of the slide Place ProPlate 16 Well Slide Module 7mm X 7mm, Delrin Snap Clips on top of the slide. Make sure it aligns with the printed blocks Place slide with gasket on its corresponding well in the 4Well Plate Repeat process for each slide to be used 7.2 BLOCKING STAGE Place a 50mL Reservoir Reagent on the bench Pour proper amount of Blocking Solution (Based on the total volume to be used)

4 7.2.3 Using a Multichannel Pippetor (200uL), pour 200uL of Blocking Solution in all the gasket s 16 wells covering the slide Repeat process for each slide to be used Proceed to shake on Orbital Shaker for 2.0 hours 7.3 SAMPLE PREPARATION Take the amount of Falcon 96-Well Library Storage Plates, Polypropylene equivalent to the amount of sample plates for the MiniChip run in CDI-LIMS Label each plate (permanent marker) in such a way that samples may not be confused (E.G. Plate 1 BARCODE, Plate 2 BARCODE, etc.) Proceed to place 0.2mL Polypropylene PCR Tube Strips on each Falcon 96-Well Library Storage Plates, Polypropylene until an exact copy of the CDI-LIMS plate to be sampled is created Determine the amount of Blocking Solution needed for a 1:12 dilution of all samples Calculate the amount of ANTI-tag (His and/or GST) needed for the total Blocking Solution volume (Final Concentration [1mg/mL]) Prepare the Sampling Solution (Blocking Solution + ANTItag)

5 7.3.7 Add 180uL from Sampling Solution to all 0.2mL Polypropylene PCR Tube Strips (Rows A-G) and 200uL for Row H Add 20uL from sample plate to dilution plate. Ensure correct matching as per original coordinates. 7.4 SAMPLING STAGE ***Avoid condensation or dryness at all times*** Bring 2-3 Nunc Rectangular 4-Well Dish Plates with previously blocked arrays to the sink area Remove cover for (1) of the Nunc Rectangular 4-Well Dish Plates Proceed to invert all arrays in such a way that all Blocking Solution is removed, but no solution is exchanged between wells Once 2-3 of the Nunc Rectangular 4-Well Dish Plates with blacked arrays have been removed from blocking Solution, proceed to bring them to the bench area and quickly refill each slide with the corresponding diluted sample, according to slide coordinates Repeat steps until all slides are still in blocking solution Bring all Nunc Rectangular 4-Well Dish Plates to Orbital Shaker for hours 7.5 SECONDARY ANTIBODY SOLUTION PREPARATION ***Photosensitive Reagent, keep out of light reach*** Secondary Antibody Solution is prepared with blocking solution (refer to step 6.1) Depending on final volume to be prepared, use a 50mL Centrifuge Tube or 250mL Receiver Wrap all the surface of the 50mL Centrifuge Tube or 250mL Receiver in Aluminum Foil Perform all necessary calculations for addition of 3 m L of Secondary Antibody Solution to each slide Cy3 Streptavidin will be added in a 1:1000 dilution (1/3) Alexa Fluor 555 Goat anti-rabbit IgG (H+L) will be added in a 1:1000 dilution (2/3) Alexa Fluor 647-conjugated AffiniPure F(ab )2 Fragment Goat Anti-Mouse IgG (H+L) will be calculated for a 1:800 dilution

6 in solution Prepare Secondary Antibody Solution in the Aluminum Foiled container 7.6 WASHING STAGE Bring 2-3 Nunc Rectangular 4-Well Dish Plates with arrays to the sink area Remove cover for (1) of the Nunc Rectangular 4-Well Dish Plates Proceed to invert all slides in such a way that all Sampling Solution is removed, but no solution is exchanged between wells (Sample Discard) Pour TBS-T solution with a constant flow in such a way that all wells for the slides contained in the Nunc Rectangular 4-Well Dish Plate become half filled Proceed to invert all slides in such a way that all Blocking Solution is removed, but no solution is exchanged between wells. Repeat (3) times After three buffer exchanges and final inverting, refill with TBS-T solution (slide cannot be left empty) Repeat for each Nunc Rectangular 4-Well Dish Plate with arrays Allow for a 5-10 minute incubation period on Orbital Shaker Repeat steps

7 Allow for a 5-10 minute incubation period on Orbital Shaker Repeat steps Allow for a 5-10 minute incubation period on Orbital Shaker 7.7 SECONDARY ANTIBODY After last washing incubation, repeat steps Remove gaskets for all arrays in the Nunc Rectangular 4Well Dish Plate Proceed to add Secondary Antibody Solution 3mL per slide Wrap all the surface of the Nunc Rectangular 4-Well Dish Plate in Aluminum Foil (Nunc Rectangular 4-Well Dish Plate can be bulk packed) Repeat steps until all arrays have Secondary Antibody Solution, cover in Aluminum Foil once assays have solution added Allow for gentle shaking on the Orbital Shaker for hours 7.8 WASHING STAGE Bring 2-3 Nunc Rectangular 4-Well Dish Plates with arrays to the sink area Remove cover for (1) of the Nunc Rectangular 4-Well Dish Plates Use the vacuum pump station to remove all Secondary Antibody Solution

8 Note: Use suction on the bottom on the array (as shown above) Pour TBS-T solution with a constant flow, making sure pouring occurs in the barcode area of the chip, in such a way that the arrays in the Nunc Rectangular 4-Well Dish Plate are covered completely (5mL). After TBS-T Solution has been added, slightly lift each slide by one side in order to wash the back part. This will diminish background during the scanning process Remove solution by carefully inverting. Repeat washing process 3 times After last solution removal, refill with TBS-T Solution (slide cannot be left empty) Repeat for each Nunc Rectangular 4-Well Dish Plate with arrays Allow for a 5-10 minute incubation period on Orbital Shaker Repeat steps Allow for a 5-10 minute incubation period on Orbital Shaker Repeat step Proceed to wash each Nunc Rectangular 4-Well Dish Plate with ddh2o Repeat (3) times. Volume must be added on barcode area. 7.9 SLIDE PROVISIONAL STORAGE Search for a Fisherbrand Microscope Slide Box Place a small piece of Paper Towel into the slide box. Start to transfer all slides to the Fisherbrand Microscope Slide Box. Make sure they retain the proper test order. All slides will be placed in such a way that the barcode will be in the right, facing front. Slide 1 will be placed in the upper box slot.

9 7.9.3 If multiple Fisherbrand Microscope Slide Box are in use, please label each box to avoid confusion in slide order Place the Fisherbrand Microscope Slide Box in the Centrifuge Slot Centrifuge at 700 rpm for 5-7 minutes Retrieve box Remove Paper Towel Proceed to scan 8.0 SCANNING PROCEDURE Open GenePix Pro 6.1 Software Locate MiniChip Images Record on computer Create MMDDYY folder Create IMAGES, SETTINGS, GPR and RESULTS Subfolders Place Slide into GenePix 4000B Scanner Open Hardware Settings on GenePix Pro Press Auto-PMT Function Save Image in IMAGES folder under corresponding test date, following the BARCODE_YYYY-MM-DD_NUMERIC SUFFIX format. For NUMERIC SUFFIX, use slide order (E.G. First Slide = 0001, Second Slide = 0002, etc.) Drag corresponding GAL file into GenePix Pro 6.1 program Align GAL File with blocks and features Use F5 (Auto-align features and blocks) Observe all blocks for interactions If an interaction is observed, using Feature Mode draw a rectangle around the pair of observed hits. Flag Good (Press O) For statistical purposes, pick two more pair of interactions within the same block and repeat Save Settings in SETTINGS folder under corresponding test date, following the BARCODE_YYYY-MM-DD_NUMERIC SUFFIX format. For NUMERIC SUFFIX, use slide order (E.G. First Slide = 0001, Second Slide = 0002, etc.) Press Analyze (Alt+A) button Save GPR file in GPR folder under corresponding test date following the BARCODE_YYYY-MM-DD_NUMERIC

10 SUFFIX format. For NUMERIC SUFFIX, use slide order (E.G. First Slide = 0001, Second Slide = 0002, etc.) Take out slide from scanner and place on provisional storage box with barcode facing left-front Repeat steps until all slides have gone through the process