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- Always read and follow the product instructions. The information contained in the kit manual is your first source of knowledge for your particular assay. If there is any special handling information that you need to be aware of, it will be recorded in the kit insert. The insert has been written to allow you the maximum amount of useful information in one convenient location. NOTE…Do not use the same kit insert repeatedly since the kits and inserts are periodically improved. The information that comes packaged with each kit contains information about any improvements and is the most current information available
- Check the calibration of the pipets that will be used for the assay. Each manufacturer should supply the recommended calibration protocol and performance specifications with each instrument. You need to verify that the volumes you are measuring are correct.
- Do not mix components from different lots or kits. Each component is lot specific and designed to give you a defined level of performance when used before the expiration date.
- Always store the kit components at the recommended conditions. In some instances, special components may need to be frozen for maximum stability. Each component will have specific storage information printed on its label. Kits containing materials that require special storage should be clearly marked on the outside of the box.
- Do not use the kit past the expiration date marked on the box. While many of the individual components may have expiration dates that extend beyond the kit date, the total kit expiration date has been determined based on the stability of all the materials stored at the suggested conditions.
- Allow the plate to warm to room temperature before opening. The wells in the microtiter plate are coated with an antibody solution. While this coating is stable and robust, moisture will corrupt it causing a decrease in performance. The coated plate is shipped in a reusable mylar bag containing a mini-desiccant packet to ensure dry conditions. When not in use, the wells should be stored at 4°C, tightly sealed in the mylar bag with desiccant. Do not throw the plate frame away after your first assay. You will need it for the remaining wells stored in the foil bag.
- Allow all reagents to warm to room temperature before opening unless instructed otherwise. Many of the reagents contain temperature-dependant components that may come out of solution when cold. Using the reagents at room temperature ensures that you have a consistent formulation from assay to assay.
- Pre-rinse pipet tips with the reagent before transferring a volume. To pre-rinse, insert the end of the pipet tip just under the surface of the reagent and draw up a volume according to the pipet manufacturer's directions using a slow even motion. Expel the volume using the same steady motion. Typically you should pre-rinse the tip three times, using the fourth “draw” as your transfer volume. This is good lab practice regardless of the tip manufacturer's claims of low retention. Precision and accuracy will be maximized by taking the time to pre-rinse tips on a consistent basis. You need to pre-rinse a tip only once if you are using it to make multiple transfers. Always change the pipet tip if it has been accidentally contaminated.
- Always use a fresh pipet tip for each standard, sample or reagent. Do not use the same tip for your standards even when pipetting from the lowest to highest concentrations. You will sacrifice precision while saving less than a penny.
- Pipet the first reagent into the bottom of each well. Pipet subsequent volumes in different locations on the sides of each well, being careful to avoid cross-contaminating reagents. A simple way to avoid contamination is to pipet one reagent into all of the wells at the same location (eg.: left side wall), then turn the plate 90° and pipet the next reagent at the same position (eg.: left side wall). Because you rotated the plate, this position will put your pipet tip at a new location within the well.
- Be careful handling the microtiter wells and reagents to prevent contamination. You have a variety of enzymes on your skin including proteases and endogenous alkaline phosphatase. If you accidentally touch a pipet tip, you can transfer these enzymes to your reagents resulting in unusable kit components. This is especially important for your substrate. The use of contaminated substrate will result in a less sensitive assay.
- If culture media (CM) samples are being analyzed, then the standards should be diluted in non-conditioned media. Do not assume that all CM are the same. Not only do the media differ in formulation, but serum supplements will also differ by type and lot. You should expect to find a basal level of endogenous analyte in media containing serum supplements. In addition, other medium supplements can contribute to the overall detection in an assay. If non-conditioned media is used as the standard diluent, then all samples will be relative to the standard curve. Using non-conditioned media usually results in a modest depression of the standard curve relative to standards diluted in kit assay buffer. This depression is acceptable since it is more important for the samples to be relative to the appropriate standard curve.
- It is simply good lab practice to run duplicates for repeatability verification. In addition, you maximize your results when you run all wells in duplicate. It is easier to edit one outlying data point than to run the experiment over again because you were not able to get an appropriate measurement for some standard or sample. An outlying measurement can result from an improper amount of a reagent being added to a well or losing a portion of the volume during preparation or incubation. While one never thinks that this will happen to them, unforeseen things sometimes happen with unfortunate results.