Western blot dilution curve

Antibody dilution in your experiments relies on many factors. It is important to understand the manufacturer's recommended dilutions and how to optimize the antibody for your own system.

Contributed by Amy Archuleta

When shopping for an antibody, most vendors supply a list of their antibody’s attributes: everything from species, to concentration, to clone. A recommended dilution is often one of the reported values. What does this number mean? How absolute is it?

What does “recommended dilution” really mean?

The recommended dilutions that are on an antibody product’s data sheet are just that – recommendations. Not guarantees. Not promises. They are suggestions based on the manufacturer’s experience, protocols, imaging systems.

Where did these dilution numbers come from?

Antibody manufacturers who have optimized their product will provide a recommendation based on their experimental results. It is important to note that these dilutions are what worked well for THEM in their lysates or cells, in their experimental conditions, in their buffers. That means that their optimal dilution may not be the same as yours in your lysates, your experimental conditions, and your buffers.
If an antibody manufacturer has not optimized the antibody in a certain application, they may report the dilution used by other scientists or collaborators who have published with the antibody. Again, this is what worked in their system, and it may be different in yours.

What does this mean for your experiments?

It means that these recommended dilutions are a great starting point as you begin to optimize the antibody in your system. However, it is important to run your own dilution titration in your experimental conditions. This will establish a signal-strength comparison of several dilutions of the antibody on either side of the recommended dilution. For example: if the recommended dilution is 1:1000, run a series of dilutions at 1:200, 1:500, 1:1000, 1:2000, 1:4000.
We recommend running your own dilution curve EVERY TIME you begin working with a new antibody, and every time you work with an antibody in a new system or experimental conditions.

What can affect the signal strength so drastically?

There are many factors that can influence antibody binding and/or signal strength: the antibody of course, but also incubation times, pH, temperature, abundance of the protein in your lysate, antibody affinity and avidity, background bands, the species in which you are testing, the age of the animal from which your tissue was harvested, even the blocking reagent you use. That is why it is worth your time to determine what works in your system BEFORE you spend hours, dollars, and mental sanity on blots or staining experiments that give inconclusive results due to lack of signal or blown-out signal.

What about different lots of the same antibody? Do I have to check every time?

To provide a short answer: yes. There is much discussion regarding how monoclonal and polyclonal antibodies factor into reproducibility. Regardless of antibody type, it is still worth your time to compare a new lot of an antibody to an older lot that you have had in your lab. There may have been degradation of the antibody in your old vial due to age or storage conditions. It is important to know that before beginning additional experiments.

What about polyclonals and reproducibility?

In addition to the possibility of degradation that can affect any type of antibody as described above, polyclonals can have other variables to consider. Quite often, different lots of polyclonal antibodies can vary incredibly due to:

  1. The possibility that they may come from different animals or
  2. Differences between blood draws over time from the same animal.

This makes it even more important to optimize every vial of antibody you receive BEFORE starting additional experiments. PhosphoSolutions has taken steps to address these very issues with polyclonal reproducibility.

Polyclonal Serum Pooling Ensures Reproducibility

At PhosphoSolutions, we pool all of our positively screened bleeds for each antibody, which eliminates many of the issues associated with the reproducibility of polyclonal antibodies. This means that every lot of our polyclonal antibodies is purified from the exact same starting material, ensuring lot-to-lot consistency. Read more about our serum pooling initiative here.

Should “Recommended Dilution” affect your decision to purchase the product?

Consider the recommended value as a starting point and one of the factors to consider in your purchase. If the recommended dilution is low – for example, 1:10 – then you will want to consider the amount of antibody that you may need to run an experiment if this suggestion is close to the dilution that works in your system. For higher recommended dilutions – for example 1:2000 – the dilution range can probably be determined and experiments run using much less antibody. Just remember your optimal dilution may be different than the recommended value.

Take home message

Keep in mind that a manufacturer’s recommended dilution is simply a place to start for your own experiments. Running your own preliminary dilution curve for each antibody in your system will save a lot of frustration down the road!

Further Reading

SDS-PAGE Demystified – The Science Behind All Those Bubbles!

Antibodies in Western and IHC

Antibody Variability

Are Monoclonal Antibodies Really More Specific?

1% SDS is the Lysis Buffer of Choice for Most Western Blots

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