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  • Antibody basics training 
    Part 4: troubleshooting


    Welcome to our training series on how to choose and use antibodies. Here we’ll guide you through topics such as selecting the right antibodies for your needs, handling and storing antibodies, antibody validation, and troubleshooting when things go wrong.

    Even the best-laid plans can go awry. And when they do, you want to have some tried and tested steps to troubleshoot your experiments. As immunoassays are so varied, it’s difficult to generalize, so to speed things up, simply select your application and get the troubleshooting guide you need.


    Part 4 overview

    4.1 Western blot troubleshooting
    4.2 IHC and ICC troubleshooting
    4.3 Flow cytometry troubleshooting


    4.1 Western blot troubleshooting

    This detailed guide takes you through some of the most common problems, like no signal, high background and multiple bands, and what you can do to fix them.

    Fix your western blot

    We also have two short videos that explain how to solve your no staining and high background issues and offer some examples of other common problems from our lab.


    Troubleshooting high background and no signal


    Troubleshooting examples from our lab



    4.2 Immunohistochemistry (IHC) and immunocytochemistry (ICC) troubleshooting

    Whether you’re dealing with tissues or cells, you can run into several problems. Here are our guides to both IHC and ICC that go through numerous causes of problems, such as no staining, non-specific staining, and high background.

    Troubleshoot IHC and ICC

    If you want to find out how to solve these common problems, our two short videos highlight the major causes and what can be done to address them.

    Troubleshooting in IHC



    Troubleshooting in ICC



    4.3 Flow cytometry troubleshooting

    Flow cytometry can be daunting to new users, especially when things don’t go according to plan. To make everything that much smoother, our guide covers the top 7 problems and how to overcome them.

    1. No signal/weak fluorescence intensity

    Signal not correctly compensated
    Check that the positive single color control is set up correctly on the flow cytometer, gated, and compensated correctly to make sure you capture all the events.
    Insufficient antibody present for detection
    Increase the amount/concentration of the antibody.

    Intracellular target not accessible
    Check if the target protein is intracellular or if the antibody epitope for a membrane protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent the internalization of cell surface proteins, all protocol steps must be performed on ice or at 4°C, using ice-cold reagents, to stop all cell reactions.

    Adding sodium azide to your experimental reagents will prevent the modulation and internalization of surface antigens, which can produce a loss of fluorescence intensity. For staining of adherent cell lines, trypsin can often induce internalization of cell surface proteins, and more gentle detachment methods may be required.

    Intracellular staining – fluorochrome conjugate too large
    Fluorochromes for intracellular staining experiments should have a low molecular weight. Large molecular weight fluorochromes can reduce antibody motility and prevent the antibody from entering the cell to label your protein of interest.

    Lasers not aligned
    Ensure the lasers on the flow cytometer are aligned correctly by running flow check beads and adjusting the alignment if necessary. If the lasers do not align correctly or if drift occurs, you may need to consider having the machine serviced.

     Target protein not present/expressed at a low level
    Ensure the tissue/cell type you are analyzing expresses the target protein and that it is present in a high enough amount to detect. 

    Soluble/secreted target protein
    Is the target protein soluble and secreted from the cell? Your target needs to be membrane-bound or cytoplasmic to be detected easily by flow cytometry. A Golgi-block step, such as with Brefeldin A, may improve the signal achieved for intracellular staining.

    Offset too high/gain too low
    Include a positive control to set up the flow cytometer correctly, using the offset to ensure the fluorescent signal from cells is not being cut-off, and increase the gain to increase the signal (within reason – care should be taken).

    Fluorochrome fluorescence has faded
    An antibody may have been kept for too long or left out in the light. The fresh antibody will be required.

    The primary antibody and the secondary antibody are not compatible
    Use a secondary antibody that was raised against the species in which the primary was generated (eg primary is raised in rabbit, use anti-rabbit secondary).

    2. High fluorescence intensity

    Antibody concentration too high
    This will result in high, non-specific binding or very high fluorescence intensity. Reduce the amount of antibody added to each sample.


    Excess antibody trapped
    This can be a particular problem in intracellular staining where large fluorochrome molecules on the antibody can be trapped within the cells. Ensure adequate washing steps and include tween or triton in wash buffers to maintain cell permeabilization.

     Inadequate blocking

    Add 1-3% blocking agent into your antibody mix, as well as a blocking step.

    3. High background/high percentage of positive cells

    Gain set too high/offset too low
    Use the positive control to set up the flow cytometer correctly, using the offset to reduce background signal from small particles and reduce the gain to decrease the signal.

    Excess antibody 
    Decrease antibody concentration. Detergent can also be added to the wash buffers to ensure any excess antibody is washed away.

    4. Two or more cell populations observed when there should be just one

    More than one cell population present expressing target protein
    Check the expected protein expression levels in the cell types contained in the sample and ensure adequate cell separation if necessary.

     Cell doublets present
    Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. Mix the cells gently using a pipette before staining and again before running on the cytometer. Cells can also be sieved or filtered to remove clumps (30 μl Nylon Mesh).

    5. High side scatter background (from small particles)

    Cells lysed
    Ensure cells in the sample have not been lysed and broken up. Samples should be fresh and prepared correctly. Do not centrifuge cells at a high rotor speed or vortex too violently.

    Bacterial contamination

    Ensure the sample is not contaminated. Bacteria will autofluoresce at a low level and result in a high event rate.

    6. Low event rate

    Low number of cells/ml
    Run 1x106 cells/ml. Ensure cells are mixed well (but gently).

    Cells clumped, blocking the tubing
    Ensure a homologous single-cell suspension by gently pipetting several times before staining. Ensure you mix again before running. In extreme cases, cells can be sieved or filtered to remove clumps (30 μl Nylon Mesh).

    7. High event rate

    High number of cells
    Dilute to between 1x105 and 1x106 cells/ml.

    Summary

    And that concludes the Antibody basics series. We hope that you’re now better equipped with the tools to take your research to the next level. As you can see, if you take the time to properly select an antibody that suits your needs, validate that it works in your setup, implement robust controls, and run even just a little bit of optimization, you’ll achieve better, more reproducible results. You’re also likely to avoid the most common issues and, therefore, hopefully, not even need our troubleshooting guide!

    If you feel ready, try taking our antibody basics quiz to test your new knowledge.

    Be sure to keep an eye on the Abcam training page as new training series for different applications become available.