Antibody basics training 

Part 2: storage and handling

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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.

In part 2 of our training series, you’ll be learning how to properly store and handle your antibodies. With proper storage and handling, most antibodies should retain activity for months, if not years.

Please remember to always refer to the manufacturer’s datasheet for specific storage recommendations.

Part 2 overview

2.1 Handling your new antibody
2.2 Storing conjugated antibodies
2.3 FAQs

2.1 Handling your new antibody

As soon as you receive your new antibody, take the following steps:

Spin
First things first: centrifuge your vial of antibody when you receive it. It has been moved around during delivery, so there’s going to be liquid in the vial cap. A quick blast in the centrifuge (10,000 x g for 20 seconds) will make sure all your precious antibodies are at the bottom of the vial – fully accessible.



Aliquot
You don’t want to take the whole vial in and out of the fridge or freezer when you use it, so aliquoting to at least 10 µL is a good idea. This way you only take out what you need on any given day. The smaller the aliquot, the more the stock concentration is affected by evaporation and adsorption of the antibody onto the surface of the storage vial, so stick to 10 µL as the minimum.

To prevent microbial contamination, sodium azide can be added to your antibody preparation to a final concentration of 0.02% (w/v). It’s worth pointing out that if staining or treating live cells with antibodies, or if using antibodies for in vivo studies, do not use sodium azide. This antimicrobial agent is toxic to most other organisms as it blocks the cytochrome electron transport system.

Learn when sodium azide needs to be removed from antibody solutions and how to do this with our sodium azide removal protocol.



Store
When it comes to storage, remember that repeated freeze/thaw cycles can denature an antibody, causing it to form aggregates that reduce its binding capacity. Because of this, make sure aliquots only go through one free-thaw cycle, with any remainder kept at 4°C. You should store your antibodies at -20°C as there’s no significant advantage to storing at -80°C. In most cases, storage at 4°C upon receipt of the antibody is acceptable for one to two weeks.

Make sure not to use a frost-free freezer: it’s unlikely your lab would, but the cycling between freezing and thawing should be avoided. For the same reason, antibody vials should be placed in an area of the freezer that has minimal temperature fluctuations, for instance, towards the back rather than on a door shelf.

2.2 Storing conjugated antibodies

Conjugated antibodies often need a little bit of extra attention since they’re more complex than non-conjugated antibodies. Here are a few pointers for proper conjugate antibody storage and handling.

	Fluorescent labels, eg Alexa Fluor®, Dylight®, FITC, PE	HRP
Handling	Aliquot upon delivery Avoid freeze/thaw cycle Store in the dark	Aliquot upon delivery Avoid freeze/thaw cycle Store in the dark
Aliquoting	Aliquot away from a direct light source When you receive the antibody, centrifuge at 10,000 x g for 20 seconds Aliquot after gently mixing with a pipette. Repeat 3-4 times. Do not mix by inversion as this may cause the solution to foam	Aliquot away from a direct light source When you receive the antibody, centrifuge at 10,000 x g for 20 seconds Do not add sodium azide to HRP-conjugated antibodies since this preservative inhibits HRP activity
Long-term storage	Follow the manufacturer's datasheet recommendations Store at -20°C Storing in amber vials or tubes covered with foil	Follow the manufacturer's datasheet recommendations Store at -20°C if it contains a cryoprotectant (eg glycerol)* Storing in amber vials or tubes covered with foil
Short-term storage	Store at +4°C short term (1-2 weeks)	Store at +4°C short term (1-2 weeks)

*Freezing and thawing enzyme-conjugated antibodies will reduce enzymatic activity in addition to affecting the antibody binding capacity. Therefore, enzyme-conjugated antibodies should not be frozen at all and should instead be kept at 4°C, unless an antibody contains a cryoprotectant and its stability has been validated for long-term storage at -20°C.

2.3 Storage and handling FAQs

In the following video, we address common questions on storing and handling antibodies.

Get answers to our dry ice FAQs.

Summary

In case you missed anything, here's a quick rundown: 

• Centrifuge your new antibody at 10,000 x g for 20 seconds to pull everything down
• You can keep the antibody at 4°C in the short-term (1-2 weeks)                     
• Aliquot to ≥10 µL
• Freeze-thaw the aliquot only once
• Add sodium azide only if needed and don’t use it for HRP-conjugated antibodies
• Store at -20°C for the long-term unless specified otherwise in the manufacturer’s datasheet
• Always refer to the manufacturer’s datasheet for specific storage recommendations
• Do not use a frost-free freezer
• You can keep your antibodies at -80°C but there’s no need unless specifically suggested by the supplier

And that's the end of part 2!

Next, we’ll focus on getting the most out of your antibodies. You’ll learn all about:

• Antibody validation
• Choosing controls
• Optimizing your experiment
• Useful step-by-step protocols

Go to Part 3.