ELISA Protocols

Posted by Jack on November 24, 2022
Table of Contents


    ELISA is a powerful technique for studying interactions between proteins. It's a simple process, but there are certain steps that you need to follow in order to get good data. In this article I'll explain how to set up an ELISA plate so that your experiment goes smoothly and gives you accurate results!

    Prepare the reagents.

    • Prepare the reagents.
    • In a separate container, prepare the following solutions:
    • Sample buffer (50 mM Tris HCl, pH 8.0; 150 mM NaCl; 0.02% Tween-20)
    • Primary antibody reagent (Fab fragment of goat anti-mouse IgG [H+L] conjugated to DyLight 649 at 1 mg/mL in PBS) and conjugate diluent (conjugate diluent is antigen-free PBS with 0.05% BSA and 0.02% Tween 20). The antibody must be prepared fresh on the day of use due to its short shelf life; however, it can be stored for up to 1 week at 4°C if stored in the presence of protease inhibitors at a concentration of 1 μg/mL or higher (e.g., 50 μL per mL).

    Prepare the plates

    Use a sterile pipette to dispense the sample.

    Use the same sterile pipette to dispense the buffer.

    Use a different sterile pipette to dispense the conjugated secondary antibodies, if needed.

    Use yet another sterile pipette to dispense primary antibodies, if needed.

    Prepare samples.

    Sample preparation is an important step in the ELISA protocol. The sample must be prepared before it can be tested, and many factors affect the quality of results. For example, if you fail to prepare your sample properly, you may get an inaccurate result that can negatively impact your experiment.

    First things first: check if your kit includes a sample preparation reagent kit or not (it will say on its packaging). If so, use this kit to prepare your samples; otherwise follow these steps:

    • Weight out 100 mg of each sample and transfer them into separate tubes using a multi-channel pipette or by using individual 10 mL rubber septa flasks for each sample (this helps prevent cross contamination).
    • Add 1 mL of deionized water (DIW) per 100 mg weight added above and mix well with vortex mixing for 30 seconds. Allow these suspensions to stand at room temperature until solid particles settle down at the bottom of the tube; discard supernatant/slurry liquid off bottom by inverting tube several times while holding upright against light source.

    Note: You may also want to add Tween 20® when preparing RBCs or FFPE tissue sections as they tend not to settle as easily due to their high protein content relative compared with other types of cells which tend have more fat content than proteins making them easier suspended in solution longer term storage requirements prior

    Add to plate.

    • Add to plate. Now that the diluent is on the plate, you can add samples to it. Using a micropipette, transfer your samples into each well and make sure not to overfill them! This can cause the sample to dry out in transit and give you inaccurate results.
    • Seal with tape or put on lids (if necessary). Once all of your samples are added, seal up your plates with tape if necessary or put on lids if you used them for storage during processing

    Wash plates.

    • Wash plates

    Wash all of the microtiter plate wells with several changes of wash buffer. Use a gentle stream of buffer to rinse each well, then allow them to air dry before proceeding.

    Add primary antibodies to plate.

    Add primary antibody to plate. Primary antibodies are used to detect antigens in your sample, and can be purchased from a range of suppliers. It is important that you store these at 4 degrees Celsius and dilute them according to the manufacturer's instructions (usually 1:50).

    Add conjugated secondary antibodies to plate.

    The secondary antibody is specific for the primary antibody, and should be conjugated to a fluorescent molecule. Examples of common secondary antibodies include Alexa Fluor™ 488 goat anti-rabbit (for use with Alexa Fluor® 488 donkey anti-mouse), Alexa Fluor® 647 goat anti-rabbit (for use with Alexa Fluor® 647 donkey anti-mouse), and Cy3 goat anti-mouse (for use with Cy3 donkey anti-goat).

    Secondary antibodies that react with both primary antibodies can also be used; however, if you are using this type of secondary antibody instead of one that is appropriate for your experiment, it's important to verify that your detection method will work with this particular set up before proceeding.

    Read the plate in a reader.

    After the plate has been washed and dried, you're ready to read it. There are two different ways to read your ELISA plate: absorbance or fluorescence.

    • Absorbance reading is an older method that measures the optical density of your sample at a specific wavelength (usually 450 nm).
    • Fluorescence readings are more accurate than absorbance readings, but they require expensive equipment and a trained eye to distinguish between weak signals.

    ELISA is a really useful technique for looking at protein interactions

    ELISA is a technique that uses antibodies to detect and measure the presence of a specific protein. ELISAs can be used as either qualitative or quantitative assays. In general, an ELISA is used to detect antigens (for example, antibodies), but ELISAs are also used for detection of other molecules (such as enzymes) by conjugating them with antibodies that recognize the molecule of interest.

    ELISAs are very sensitive because they use a secondary antibody that has been labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase (AP).


    ELISA is a really useful technique for looking at protein interactions. It allows us to see how much of each protein is present, and then work out whether this correlates with disease states or not. The protocol outlined here should be followed when performing an ELISA experiment in order for it to be successful.

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