Take the plunge when pipetting, but be sure to use calibrated pipettes and to prime them before ejecting the sample.Credit: Urbazon/Getty
It takes time to get the hang of new immunodetection methods, and during this learning period you may well see your share of failed experiments. Christiana Binkley, a PhD student in cellular and molecular biology at the University of Wisconsin-Madison, recalls one notable flop in her early experimenting days. She was using western blotting to study large ribosomal proteins, employing a protocol with two overnight steps. “Finally, I had that blot, and went to image it, but there was nothing there. It was just the biggest disappointment ever.”
Binkley, and other ‘immunodetectives’, have learned from their investigations. Their simple advice on optimizing protocols can help minimize risk of disappointment when it comes to antibody-based techniques. It’s a process of trial and error, but there’s a systematic way of doing it.
Understand the science behind the technique
Getting to know the scientific principles behind the method empowers you to make purposeful protocol adjustments. For example, if you’re using western blotting to analyse protein levels, it’s worth knowing that large proteins take longer to travel through gel than small ones, and that the acrylamide percentage in the gel determines which size proteins you can separate — a lower percentage is better to separate larger proteins. With this information, you can make a decision about the type of gel needed, and for how long you should run it.
Immunodetection techniques are based on the principle of a primary antibody recognizing an epitope of the target protein, and a secondary antibody detecting the primary antibody. To get a clear signal, the primary antibody needs to specifically recognize your protein. If you’re using immunofluorescence or immunohistochemistry to stain cells or tissues, it may be a good idea to run a western blot first to make sure the antibody detects the correct protein and is not creating off-target signals.
Not all primary antibodies work for all assays. The primary may not recognize the epitope if it’s in a different conformation. Some antibodies only recognize denatured epitopes, such as those used in western blots; some only recognize native epitopes, such as those detected by flow cytometry. Some recognize both.
Which secondary antibody you use depends on the species in which your primary antibody was produced, but there are a few other things to keep in mind. For example, if you’re doing a multiple labelling experiment, you can avoid off-target effects by using a secondary antibody specific to one IgG subclass, or a cross-adsorbed secondary antibody with minimal cross-reactivity. You’ll also need to consider the method you’re using. With immunofluorescence microscopy, your secondary antibody will need to be labelled with a fluorophore that corresponds to the filters on your microscope.
Consider the anatomy of your sample, and where in the cell or tissue you expect an antibody to bind. Federica Franciosa, a physiology PhD student researching neuronal plasticity at the University of Bern, describes an easy test to determine whether antibody staining is picking up the correct target in her neuronal cells. “If I know that a protein marker should show up in the dendrites, and I see staining in the soma, I would not trust that.”
Double-check the equipment
Each immunodetection method uses its own set of tools, from electrophoresis setups to multi-well plates. Every time you learn a new technique, or do a familiar experiment but in a new lab, test your equipment. Don’t take anything for granted.
Franciosa once prepared mouse skin samples in optimal cutting temperature (OCT) compound for immunostaining, but then used the wrong type of slide. “I didn't use the slides that sealed in the slice,” she explains, “so when I did the incubation steps, my sample was swimming around.”
Pipettes are important too, as Adam Kensinger learned. Before starting his PhD at Duquesne University in Pittsburgh, Pennsylvania, he did an internship at secondary antibody manufacturer Jackson ImmunoResearch, testing new secondary antibodies. Even though he used a robotic pipetting machine to carry out large-scale ELISA tests, Kensinger occasionally needed to work with manual pipettes – and be just as accurate as the machine. “It's really important to have calibrated pipettes,” says Kensinger, “and to prime them before ejecting the sample, because that's where a lot of errors can happen without you even knowing.”
And sometimes, equipment just needs to be replaced. When Binkley started studying ribosomal proteins, she was the only person in her lab using western blotting. “My lab had a really old semi-dry transfer apparatus. It had lots of salt build-up, and no-one had used it in a decade.” She tried to get it to work, but kept struggling with the transfer step. “We ended up buying a new one — and that made a huge difference.”
When sharing a lab with other people who are also doing western blotting, it’s a good idea to have your own buffer solution.Credit: Jackson immunoresearch
Optimize dilutions, incubation and blocking steps
When using a new antibody, you will need to find your optimal dilution. If the antibody concentration is too high, you could get background signal; too low, and you might not effectively detect your target – especially if there’s only a small amount of protein in your sample. Do a few test runs using a range of antibody dilutions. With a western blot or ELISA, you can also optimize the amount of sample in each lane or well.
For Kensinger, these optimization steps were a challenging aspect of learning ELISA. One of the things he had to troubleshoot was the dilution series for the standard curve used to calculate the amount of protein in the samples. “If you don't have a properly diluted standard on the ELISA plate, that can cause issues.” Carefully calculating and pipetting the dilution series makes all the difference.
Other steps in the protocol influence the experiment’s output. You might get a cleaner background by varying the blocking buffer, incubation time or temperature — or by adjusting the shaker settings. “I found that movement was really important,” says Franciosa, about her experience with western blots. “Starting from the blocking phase, the nitrocellulose membrane had to be moving.” If it’s stationary, the blocking buffer or antibodies won’t cover the whole membrane.
Franciosa has a tip for people sharing a lab with other western blotters. “Make your own buffers,” she urges. “Because then, whatever happens, you know that you only have yourself to blame.”
Keep detailed notes
It can take a lot of tweaking to achieve the optimal protocol for given proteins and antibodies. Once you’ve found your perfect combination of conditions, you’ll want to replicate them every time. The key is to take note of everything you’ve changed.
“Be consistent, and write down how you deviated from the protocol,” says Kensinger. His time working at Jackson ImmunoResearch taught him the importance of keeping a detailed and up-to-date lab notebook, with pictures of gels and blots. “If you get an unusual result, you can see what happened.”
There are a lot of opportunities to hone the protocol for your experiment. “The art is in optimization,” says Binkley. “Trial and error are your best friends.”