EnSpire® Label-free Technology Label-free Biochemical Assay: Biotin Binding Assay Using an EnSpire-LFB Pre-Activated Sensor Plate Introduction ® This protocol describes how to perform a label-free biochemical assay on the EnSpire Multimode Plate Reader to measure the binding of biotin (244.31 Da) to the protein streptavidin (60 kDa), which is immobilized at the bottom of each well of an EnSpire-LFB 384-well plate. Materials ® Streptavidin (SA) (Sigma-Aldrich catalog #S-4762) ® Biotin (Sigma-Aldrich catalog #B-4501) ® Sodium Acetate (Fisher Scientific catalog #BP333-500) ® Hydrochloric Acid (Fisher Scientific catalog #A144) ® Sodium Hydroxide (J.T.Baker catalog #3734-01) ® 1X Phosphate Buffered Saline (PBS) (Sigma-Aldrich catalog #P-3813) EnSpire-LFB 384-well plate (Label-free enabled sensor plate for biochemical assays with aminecoupling, pre-activated) (PerkinElmer catalog #6057410) Note: Keep plate sealed in package until ready to use Equipment EnSpire Multimode Plate Reader 384-well compatible multichannel pipette (preferably 16-channel) Aspiration wand (V&P Scientific catalog #VP186LP), including external vacuum source Standard laboratory grade tape used to tape ends of aspiration wand so as to leave 10 µL residual volume in label-free plate (tape and test wand prior to assay) ® 384-well round bottom polypropylene sensor plate (Corning catalog #3657) 15 and 50 mL conical tubes Large volume pipette (5-25 mL aspirator pipette refills) Plate centrifuge Manual pipettes (10-1000 µL) Reaction tubes (1.5 mL) ® Foil plate seals (VWR catalog #BK538619 18 MΩ distilled, filtered water (or cell culture-grade) 0.22 mm cellulose acetate filter system (1 L capacity) Reagent reservoirs (100 mL) Glass or plastic storage bottles for making buffers (250–1000 mL) Assay Workflow The workflow for the EnSpire label-free biochemical binding assay is detailed in figure 1. Figure 1: Workflow for EnSpire label-free biochemical binding assay. Tips 1. When preparing label-free plates, always make up all of the solutions first and open the plate package last to minimize exposure to air. 2. Try not to touch the glass bottom of the plate. If dust/fingerprints are visible, remove with 100% ethanol and a low-lint precision wipe. 3. Always use the same source of reagents and buffers within an assay, as well as when running multiple assays on a given day. 4. Avoid creating air bubbles in the wells of label-free-plates. This is very important when adding compounds. Make sure that no air bubbles cover the sensor on the bottom of the plate. Small bubbles on the liquid’s surface are less critical. www.perkinelmer.com 2/7 5. When using the hand pipettes, be sure that bubbles are not being pulled up into the tips. Also, try to watch the fluid level in the tips as it is being dispensed to ensure that the same volume goes into each well. 6. Prime the tips of the pipettes in the solution before use. Prime the tips by aspirating and purging once with the amount of solution that will be utilized. 7. Perform a visual inspection of all plates with solution (label-free or source) to verify there is liquid in each well. Reagent Preparation 20 mM Sodium Acetate buffer, pH 5.1 1. To prepare 500 ml of sodium acetate buffer, add 0.8203 g sodium acetate to ~485 mL 18 MΩ water and stir until dissolved. 2. Adjust the pH to 5.1 using ~6 N HCl (use sodium hydroxide if too low). 3. Add the solution to a graduated cylinder and adjust the volume to 500 mL with 18 MΩ water. ® 4. Filter the solution using a Corning 0.22 µm cellulose acetate filter system. 5. Store solution at room temperature. Discard the solution if any signs of microbial growth appear during storage. Smaller quantities of acetate buffer should be prepared if assays will not be run on a 1 regular basis . 1X Phosphate Buffered Saline (PBS) 1. Reconstitute 1 packet of PBS with 1 L of 18 MΩ water. ® 2. Filter the solution using a Corning 0.22 µm cellulose acetate filter. ® Streptavidin (Sigma-Aldrich catalog #S-4762-5MG OR #S-4762-10MG) 2 1. Stock Solution: Make a 1 mg/mL stock solution of streptavidin in phosphate buffered saline (pH 7.4) by adding 5 mL PBS to each 5 mg vial of streptavidin or add 10 ml PBS to each 10 mg vial. Vortex to ensure homogeneity. Store 1 mL aliquots in a -20°C freezer. Streptavidin aliquots should not be frozen again once thawed. 2. Working Solution: Prepare a 75 µg/mL solution of streptavidin in 20 mM sodium acetate buffer (pH 5.1) by performing a 13-fold dilution of 1 mg/ml stock. Add 770 µL of 1 mg/ml streptavidin to 9.23 mL of 20 mM sodium acetate buffer. Working solutions should be freshly prepared for each assay. Biotin 1. Stock Solution: Prepare a 1 mM stock solution of biotin in PBS. Weigh out 4.2 mg of biotin in a 50 ml conical tube. Dissolve biotin in 17.2 mL 1X PBS to yield a 1 mM solution (4.2 mg Biotin = 0.244 MW 3 X 1 mM x 17.2 mL). Vortex the biotin solution until completely dissolved. 2. Working solution: Prepare 6 mL of an 80 µM working solution of biotin by diluting the 1 mM stock of biotin. Add 480 µL of 1 mM biotin to 5.52 mL of PBS and vortex solution to ensure mixing. Working solutions should be freshly prepared for each assay (within 24 hours). 1 If buffer is stored, control of pH before the assay is recommended as pH 5.1 might not be stable over time. ® Check the label carefully: Sigma-Aldrich sells #S-4762-.5MG as well. This only includes 0.5 mg streptavidin instead of 5 mg. 3 1 mL aliquots can be stored in -20°C freezer. Aliquots should not be frozen again once thawed. www.perkinelmer.com 3/7 2 Label-free Plate Preparation 1. Add 15 µL of 75 µg/mL streptavidin in 20 mM sodium acetate buffer, pH 5.1 (or 20 mM acetate buffer 4 alone) into the appropriate wells of the label-free plate (see plate map below). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 A B C D E F G H I J K L M N O P 15 µL buffer control wells (20 mM Sodium Acetate, pH 5.1) 15 µL 75 µg/mL Streptavidin in 20mM Sodium Acetate, pH 5.1 Figure 2: Plate map for assay preparation. 2. Place the lid on the label-free plate and centrifuge at 800 rpm for 1 min. 3. Incubate label-free plate at room temperature for 60-90 min (This step can also be done the day before the assay and then seal the plate and incubate overnight at 4°C). 4. Prepare aspiration wand prior to assay by taping ends so that residual volume left in plate is 10 µL. 5. Wash plate 3-4 times using 25 µL PBS with aspiration wand. a) b) c) d) Aspirate streptavidin solution from all wells of label-free plate. Add 25 µL PBS to all wells of label-free plate. Aspirate PBS solution from all wells of label-free plate. Repeat steps b & c for a total of 3-4 washes and aspirations. 6. Add 5 µL PBS to all wells to leave a final volume of 15 µL PBS in label-free plate. 4 Wells with buffer only and streptavidin in buffer can be used to calculate the immobilization rate of streptavidin on the label-free plate. www.perkinelmer.com 4/7 24 7. Place a plastic lid over the label-free plate and centrifuge at 800 rpm for 1 min. 8. Allow the label-free plate to soak in PBS buffer for a minimum of 2 hours prior to running the binding assay. The timing begins after the final PBS is added. The last 30 min of the soak can be done in the EnSpire Plate Reader to allow temperature equilibration of the label-free plate. 9. While label-free plate is soaking, prepare Biotin Binding Assay Source Plate and create the biochemical assay protocol on the Enspire Plate Reader. Biotin Binding Assay Source Plate Preparation 1. See Biotin Binding Assay Plate map below. ® 2. Add 50 µL PBS to the corresponding wells of a 384-well Corning round bottom polypropylene sensor plate (wells marked in blue and gray). 3. Add 100 µL of 80 mM biotin to wells B7-B12 and 50 µL in wells A13-H18 (wells marked in orange). 4. Add 50 µL PBS to wells marked in yellow to use for 1:2 dilutions. 5. Using a 384-well multichannel pipette, serially dilute 50 µL 80 mM biotin (wells B7-B12) into the wells shown below (wells C7-C12). Continue this serial dilution scheme until you reach wells O7-O12. 6. Optional: Centrifuge source plate at 800 rpm for 1 min. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 A B 80 µM biotin (40 µM final) C 1:2 dilutions in PBS ↓ D E F G H I J K L M N O P 50 µL PBS 50 µL biotin serial dilution (2X stock starting at 80 µM) 50 µL biotin (2X stock of 80 µM, additionally used to calculate Z' high) 50 µL PBS (used to calculate Z' low) Figure 3: Plate map for biotin compound preparation. www.perkinelmer.com 5/7 24 EnSpire Biochemical Assay Protocol 1. Open the EnSpire Plate reader’s Manager Software. 2. Create a New Protocol (Label Free, biochemical, 384 well). 3. Add a “Measurement A”. Add 5 repeats for the baseline with 160 sec interval (~14 min) and add 5 repeats for the final read with 160 sec interval (~14 min). 4. Optional: Add data analysis (e.g. response (pm), response for selected repeat number, blank correction, dose-response curve, Z’) and define calculation properties. 5. Select the whole plate for measurement. 6. Optional: Select appropriate well options for data analysis. 7. Name assay as appropriate (i.e. Biotin BCA date, initials). 8. Save assay. 9. Load plate (if not loaded already) and start baseline reads. A slight negative drift in pm is normal (~0.5 pm/repeat). 10. Once baseline read is complete, plate will automatically unload, and Biotin Assay can be performed. Biotin Assay 1. Using multichannel pipette, transfer 15 µL of contents from each well of the Biotin Binding Assay Source Plate to the corresponding wells of label-free plate (15 µL from source plate plus 15 µL on label-free plate → 1:2 dilution). Be careful not to touch the bottom of the wells of the label-free plate. 2. Mix carefully 5 times using multichannel pipette. Again, do not touch bottom of wells of label-free plate. 3. Load plate into the EnSpire Plate Reader and start final reads. You should see an increase of ~30 pm on the first repeat of the final read on high biotin concentrations. This signal should then remain stable for all 5 repeats of the final read. 4. Analyze data. -7 -7 5. Calculate respective EC50 values and Z’ values: EC50 ~ 1E – 5E M. Representative results for a biotin binding assay, showing biotin binding to immobilized streptavidin, are shown in figure 4. www.perkinelmer.com 6/7 Figure 4: Representative results of biotin binding to immobilized streptavidin. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2014, PerkinElmer, Inc. All rights reserved. PerkinElmer ® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. www.perkinelmer.com May 22 2014. 7/7