It’s not just the antibodies that matter when you’re prepping for your experiment - there are a number of outside factors that need to be considered as well. In this post, we’ll touch on antibody storage, buffer considerations, and give just the lightest of nods towards conjugates.
Storage
Once you get your exciting new antibody, the second thing you’ll want to do is store it away safely. The first thing you should do is record the manufacturer, lot number, and expiration date somewhere you can easily check it whenever you use the antibody. This information is important, and you’ll need to track it throughout your experiments.
Now that that’s out of the way, you can put your antibody in short term storage (think days to weeks) in the 4°C. If you’ll only be using it every few month or if you'll be going through small aliquots at a time, the -20°C is your best option, in aliquots no smaller than 10uL. You can leave them at room temperature when you’re working with them, but avoid freeze-thaw cycles, as they’ll degrade your antibody.
At some point in your long and illustrious lab career, you will likely accidentally leave your antibody out on the bench overnight. Do not panic! Typically, antibodies are okay if they’re left out overnight, so mark the vial, add the incident to your records, and consider the following questions as you move forward:
- Was a light-sensitive conjugate on the antibody left exposed to light?
- Has this antibody been left out, gone through a freeze-thaw-freeze cycle before, or otherwise been subject to mishandling?
- Is the experiment you’re planning to run with the antibodies time-sensitive, using precious samples, or being compared to other data in a manner that demands high accuracy and consistency?
If the answer to any of these questions is “yes,” then I would suggest either testing the antibody efficacy through a standard curve, another quick experiment, or using a different vial and saving the poor, abandoned antibody for a…less sensitive moment.
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| Fig 1: Blugene fading into darkness because they accidentally left their antibodies out. It's okay, Blugene! |
Buffers
You know what temperature to store your antibodies at, but it also matters what they’re stored in - their buffer. Buffers are made of chemicals, which have an unfortunate tendency to react with other chemicals and biological structures, some of which you may need in your experiment.
If you are planning to conjugate your antibody in the lab, you’ll therefore want to research your conjugation reaction to understand if any of your buffer components could affect the reaction. For instance, sodium azide, a common antimicrobial agent added to buffers, can bind to amine groups present in some conjugates, blocking the conjugation reaction. Many commercial antibodies come with sodium azide already included in the buffer, which will be noted on the product sheet. Thankfully, sodium azide can easily be removed from the buffer and then re-added after the conjugation reaction is done, through a variety of methods or commercial kits.
However, antibodies conjugated to horseradish protein (HRP), primarily used in Western blots and ELISAs, cannot be stored in sodium azide at all, as it inhibits HRP. In those cases, thimerosal at a concentration of 0.01% w/v can be used as an alternative antimicrobial.
Conjugates
And HRP brings us handily to the final topic of this post, the conjugates, or signaling molecules! Please keep in mind that this is just a brief introduction to the wide, wild, and fascinating field of signaling molecules.
Assay Type
Westerns and ELISAs mostly use HRP, a chemiluminescent conjugate that can be activated to emit light with a simple kit, and is readily available on any number of secondary antibodies. And look - if it ain’t broke, don’t fix it. HRP is easy to find, simple to use, not sensitive to light in its inactive state, and cheap. Unless there’s a specific reason you can’t use it for your ELISA and Western, I’d highly recommend sticking with what works.
To balance this out, however, is pretty much every other assay you can do. Most non-HRP conjugates are fluorophores, which work not by reacting to a chemical, but by emitting a specific wavelength of light in response to excitation by a laser, which is then read by a detector panel in the machine. Fluorophores are light sensitive, so antibodies conjugated to them need to be stored either in a dark, opaque vial or wrapped in aluminum foil to prevent ambient light from degrading them. You’ll also need to know the wavelength of laser needed to excite them - and it’s a good idea to make sure that laser is available in your machine before you make your purchase. Picking a single fluorophore for an experiment is easy, but what about those times you need more than one?
Multiplex assays
Multiplex assays involve multiple antibodies, each with a different signaling molecule, that all need to be detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully this is an Antibody 101 post, so we'll keep it simple. If you follow the two basic guidelines below, you’ll likely be able to create a small, usable panel.
First, fluorophores vary in brightness. You don’t want a super-bright fluorophore, like PE, on an abundant target, as you risk getting so much signal that you’ll flood out other signals. But PE can be very helpful if you're trying to find a relatively rare protein in an experiment with many proteins of interest! Research the brightness of your fluorophores before buying and try to inversely pair brightness with abundance whenever possible.
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| Fig 2: Try to inversely pair fluorophore strength with protein abundance. |
Second, you’ll need to consider spectral overlap. Fluorophores signal are actually quite broad, resulting in spectral overlap, or the signal "spilling over" into another channel's detection range. Though you can compensate for this when reading your data, you’ll want to pick fluorophores with the least spectral overlap between them whenever possible. This will allow for the most accurate read and reduce needed compensation.
Working with antibodies can get a bit complicated, but hopefully this helps you successfully select - and keep! - antibodies that are a good match for your experiments, and avoid the awkwardness of getting the right antibody with the wrong everything else.
References and Resources
References
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Boston University Flow Core. Fluorochromes Brightness Chart. Boston University Flow Core. Retrieved January 31, 2022, from https://www.bu.edu/flow-cytometry/files/2013/06/Fluorochromes-Brightness-Chart.pdf
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Syracuse Flow Core. Syracuse flow core spectral overlap. Syracuse University Flow Core Facility. Retrieved January 31, 2022, from https://flowcore.syr.edu/help/spectral-overlap/
More resources on Addgene’s blog
- Antibodies 101: Monoclonal Antibodies
- Antibodies 101: Polyclonal Antibodies
- Antibodies 101: Introduction to Antibodies
Resources on Addgene.org
- Find plasmids encoding recombinant antibodies at Addgene
Topics: Antibodies
Topics: Antibodies
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