NPTII ELISA Qualitative User Guide neomycin phosphotransferase II Catalog number: PSP 73000

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1 List of contents Lot number Item 96 wells 288 wells 480 wells 4800 wells Antibody-coated 96-well microtiter plates Peroxidase enzyme conjugate (bottles A and B) ml ml ml 5.0 ml TMB peroxidase substrate solution 25 ml 40 ml 60 ml 550 ml MRS-2 component 3 ml 6.5 ml 11 ml 102 ml Positive control *** The above items should be stored at 4 C. PBST buffer, 20X liquid concentrate, 50 ml pouches or dry powdered, 110 grams X 110 g PEB1 extraction buffer, 10X concentrate 50 ml 100 ml 100 ml 2 X 500 ml The above items should be stored at room temperature. Technical Information Thank you for purchasing the Agdia ELISA Kit, catalog number PSP This kit can be used for qualitative or quantitative analysis of NPTII in plant tissue extracts. If you have any questions about using this kit, please contact Agdia, Inc. Monday Friday by phone or by info@agdia.com. ***Note: The positive control(s) included in this kit are intended for qualitative use only. For quantitative purposes, a NPTII certified reference standard (LST 73000) is available for purchase from Agdia. A quantitative user guide is available on Agdia s website or can be ed to you upon request. 1. Sample Extraction Buffer: PEB1 is the extraction buffer. It is available only from Agdia, as a 10X concentrate. This buffer substantially improves the performance of the test, providing improved response and reduced backgrounds for many kinds of samples. 2. Positive Control: The positive control included in this kit is for qualitative purposes only. 3. Sample Incubation Time and Temperature: The test is very sensitive to ambient temperature. Sample incubations longer than 2 hours at room temperature will reduce the upper useful range limit. Decreasing sample incubation time will raise the upper and lower useful range limits. Storing the kit Store all kit components at the recommended temperature to assure their full shelf life. Each ELISA plate pouch contains a desiccant packet. Keep the plate sealed in the pouch with the desiccant and store in the refrigerator. Do not store prepared 1X buffers from day to day. Safety Prevent direct skin and eye contact with, or ingestion of, product components. Obtain medical attention in case of accidental ingestion of kit components. Always wash hands thoroughly after using this product. If you have any questions about this product, please contact Agdia. m

2 You will need NPTII ELISA Copy of loading diagram Sample extract bags, mortar and pestle, or preferred method of extraction Airtight plastic box Paper towels Distilled water Micropipette for measuring enzyme conjugate Test tube with cap, such as a screw-cap centrifuge tube Preparing for the test Prepare buffers Prepare only as much 1X buffers that will be needed for one day. PBST is supplied as either 20X concentrate or as a powder. PBST 20X concentrate PBST powder Prepare 1X PBST buffer by diluting one 20X pouch of PBST buffer with 950 ml of distilled water. Prepare 1X buffer by dissolving PBST buffer powder in distilled water according to the table below: Buffer powder Distilled water 5 g 500 ml PEB1 Prepare testwells Prepare humid box Make loading diagram Prepare enough (1X) PEB1 extraction buffer for the samples being tested. Dilute the concentrated PEB1 extraction buffer to working (1X) dilution with distilled water. If you will be using less than a full 96-well plate, remove any unused strips and seal them in the foil pouch with the desiccant. Using a permanent marker, number the strips in case a strip becomes separated from the frame. Prepare a humid box by lining an airtight container with a wet paper towel. Keeping testwells in a humid box during incubation will help prevent samples from evaporating. Make a copy of the loading diagram and record the locations of your samples and controls. We recommend that you use a buffer well and a positive control well on each plate each time you run the test. Prepare samples and controls Grind and dilute samples Reconstituted controls Grind sample with 1X PEB1 extraction buffer at a ratio of 1:10 (tissue weight in g:buffer volume in ml. You can use Agdia's sample extract bags, a mortar and pestle or other grinding devices to extract samples. If you are using a mortar and pestle, wash and rinse it thoroughly between samples. Reconstitute lyophilized positive control with 1.0 ml of 1X PEB1 and lyophilized negative control with 2.0 ml of 1X PEB1. m

3 Make control aliquots After preparing the positive and negative control, divide them into aliquots, each sufficient for one use. Dispense aliquots into tubes that can be securely capped. If you will be using a control in one well each time you run the test, prepare 120 µl aliquots. If you will be using a control in two wells, prepare 220 µl aliquots. Each aliquot should be sufficient for the tests to be run plus a small additional volume to assure easy dispensing. Control aliquots must be stored frozen (-20 C freezer or household freezer). Do not thaw until just before use. At the time of each test run, remove from storage only the aliquots that will be used. Allow the tubes to thaw and mix the contents thoroughly. Do not refreeze controls. Do not use the positive control as a standard for a quantitative assay. Test Procedure 1. Dispense samples Following your loading diagram, dispense 100 µl of prepared sample into sample wells. Dispense 100 µl of each standard into standard wells for quantitative analysis or 100 µl of reconstituted lyophilized positive control into control wells for qualitative analysis. Dispense 100 µl of PEB1 extraction buffer into buffer wells. 2. Incubate plate Set the plate inside the humid box and incubate for 2 hours at room temperature. Incubating overnight in the refrigerator (4 C) is also satisfactory, however, with long sample incubations, the upper useful range of the test will be reduced and the lower useful range including assay background may be elevated. 3. Prepare conjugate diluent To prepare enzyme conjugate diluent, mix MRS-2 component with 1X PBST buffer at a ratio of 1 part MRS-2 component to 4 parts 1X PBST buffer. The volume of enzyme conjugate diluent required depends on the number of test wells used, with 100 µl needed per test well. To estimate the volume needed, prepare 1 ml for each 8-well strip used, or 10 ml for each 96-well plate. 4. Prepare enzyme conjugate A few minutes before the incubation is complete, prepare the enzyme conjugate, using enzyme conjugate diluent and enzyme conjugate bottles A and B. You should calculate the volumes to use from bottles A and B based on the volume of enzyme conjugate diluent you will use and on the dilutions given on the bottles. For example, since the dilutions given on bottles A and B are both 1:100, and if you are preparing 1 ml of enzyme conjugate, you should first dispense 1 ml of enzyme conjugate diluent. Then add 10 µl from bottle A and 10 µl from bottle B to the enzyme conjugate diluent. Use a new, sterile pipette tip for each bottle to prevent contamination. After adding the reagents from bottles A and B, cap the container and mix by inverting it several times. It is important to mix the enzyme conjugate well. Set the prepared enzyme conjugate aside. You will need it after washing the plate. Note: Always prepare enzyme conjugate within 10 minutes before use. m

4 5. Wash plate When the sample incubation is complete, wash the plate. Use a quick flipping motion to dump the wells into a sink or waste container without mixing the contents. Fill all the wells completely with 1X PBST, and then quickly empty them again. Repeat 7 times. After washing, hold the frame upside down and tap firmly on a folded paper towel to remove all droplets of wash buffer. Inspect the testwells. All wells should be free of plant tissue. If tissue is present repeat the wash step and tap firmly on a paper towel. 6. Add enzyme conjugate Dispense 100 µl of prepared enzyme conjugate per well. 7. Incubate plate Incubate the plate in the humid box for 2 hours at room temperature. 8. TMB substrate solution TMB substrate solution should be warmed to room temperature before use. Measure out the volume needed into a clean container just after addition of the enzyme conjugate. 9. Wash plate As before, wash the plate 8 times with 1X PBST. Inspect the wells looking for the presence of air bubbles. Tap firmly on the paper towel to remove remaining wash buffer and any air bubbles. If air bubbles are still present they may be broken with a clean pipette tip. 10. Add TMB solution Dispense 100 µl of TMB substrate solution per well. 11. Incubate plate Incubate the plate for 15 minutes. 12. Evaluate results Measure O.D. s on a plate reader at 650 nm. Air bubbles which are present at the time of reading can alter results, if in the light path. Agdia recommends that bubbles be eliminated prior to reading. Wells in which a blue color develops indicate positive results. Wells in which there is no significant color development indicate negative results. Test results are valid only if positive control wells give a positive result and buffer wells remain colorless. If either control well does not show the appropriate color, please repeat the test procedure. If the problem persists, contact Agdia for further assistance. Optional: Stop solution may be added to terminate the peroxidase/tmb reaction. Add 1N HCI at 100 µl per testwell. Read at 450 nm endpoint up to one hour after addition. m

5 Buffer formulations PBST Buffer (Buffer 1X) The following buffer is a standard part of your kit. The formulation is provided for your reference. Dissolve in distilled water to 1000 ml: Sodium chloride Sodium phosphate, dibasic (anhydrous) Potassium phosphate, monobasic (anhydrous) Potassium chloride Tween g 1.15 g 0.2 g 0.2 g 0.5 g Adjust ph to 7.4 m

6 Date Test Test performed by A B C D E F G H