Antibody basics week 2: storage and handling

Welcome to our training series on how to properly choose and use antibodies. Here we’ll guide you from choosing which antibodies best suit your needs, how to handle and store antibodies through to validation, and how to troubleshoot when things go wrong.


Week 2: storing and handling your antibody

​​With proper storage and handling, most antibodies should retain activity for months, if not years. Look after your antibody and it’ll look after you! Seriously, just a few simple steps and you’ll be keeping your antibody working as intended for much longer.

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


Overview

2.1 Handling
2.2 Storing
2.3 FAQs


2.1 Handling your brand-new antibody


​​Spin

First thing’s first: centrifuge your vial of antibody when you receive it. It’s going to have been through more "activity" than usual during delivery, so there’s going to be liquid in the vial cap. A quick blast in the centrifuge will make sure all of your precious antibodies are at the bottom of the vial – fully accessible. Put the antibody at 4oC until you have a chance to aliquot – you can keep it at 4oC for up to two weeks if necessary, but you should probably aliquot right away.

Aliquot

​You don’t really want to be taking the whole vial in and out of the fridge or freezer when you use it, so aliquotting to 10 or more µL is a good idea. (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.) This way you only take out what you need on any given day.

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 well as it blocks the cytochrome electron transport system.

Sodium azide removal protocol
Learn when sodium azide needs to be removed from antibody solutions and how to do this.
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​​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. You should store your antibodies at -20oC as there’s no significant advantage to storing at -80oC. Oh, and make you don’t have a frost-free freezer! It’s unlikely your lab would, but it’s the cycling between freezing and thawing that is exactly what 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.

>>> Get all the storage details here


2.2 Storing conjugated antibodies

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




Fluorescent labels

e.g 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

Aliquotting

  • Aliquot away from 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 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

  • Store at -20°C
  • Storing in amber vials or in tubes covered with foil
  • Store at -20°C
  • Storing in amber vials or in 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)

>>> Learn the difference between different antibody conjugates


2.3 Storage and handling FAQs

We get a lot of the same questions when it comes to how best to store and handle antibodies. So, here are the answers to the top 5 questions:

2.1 Your brand-new antibody

Part 2 - Storage and Handling FAQs

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​>>>  Get answers to our dry ice frequently asked questions


Summary

In case you missed anything, here’s a quick rundown

  • Centrifuge your new antibody for 20 seconds; spin it 10,000 x g to pull everything down
  • You can keep the antibody at 4oC in the short-term
  • Aliquot to ≥10 µL
  • Freeze-thaw the aliquot only once
  • Add sodium azide if needed
  • Store at -20oC for the long-term
  • Do not use a frost-free freezer!
  • You can keep your antibodies at -80oC but there’s really no need unless specifically suggested by the supplier


And that's the end of week 2!

Next week, we focus on really getting the most out of your antibodies.

You’ll learn all about:

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

In case you missed or want to revisit the first part, you can find it here:

Week 1: choosing the right antibody

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