Western blotting is the most common method for detecting and quantifying specific proteins. The cost of running a Western is largely driven by detection antibodies, which are used to probe membrane-bound target protein(s). Although some antibodies can be produced cheaply in-house, most antibodies must be commercially ordered. Reusing antibodies for subsequent Westerns can save your lab a considerable amount of money. In this week’s Bench Tips, we investigate the boundaries of this common, but not well-documented, practice.
Many researchers routinely use fresh antibodies for every blot out of fear of losing work or sacrificing reproducibility—a method reinforced by commercial interests. But a little experimentation can quickly demonstrate that primary antibodies are highly amenable to recycling. If you’re doing this for the first time, you’ll want to be aware of some important factors, discussed below.
Storage limitations
The biggest risk in reusing primary antibodies is microbial contamination. Antibodies for Western blotting are often diluted in solutions of 5% skim milk, ranging from a 1/100 to 1/500,000 dilution from a 1 mg/mL stock solution. Microbial growth is a big concern when storing your primary antibody dilution at 4°C for re-use on later blots. To avoid unwanted microbes, you can add sodium azide (0.02%). (Although most antibodies are packaged with sodium azide in the buffer, there’s not enough to be effective in larger diluted volumes.) Never include sodium azide when conjugating your antibody to a horseradish peroxidase (HRP), however, as it can inactivate peroxidase and quench signal downstream. Also, sodium azide is toxic, so work under a fume hood.
Despite the addition of sodium azide, milk eventually will go bad. It’s therefore not worth reusing this antibody solution more than three or four times. (Your nose will know when spoilage starts to kick in.) As an alternative, diluting in 5% BSA solution is a good option. BSA functions as a protein stabilizer, allowing you to reuse antibodies longer when storing in the lab fridge and working with HRP.
If you need to reuse your antibodies over an indefinite period of time between blots, you can make up the dilution in skim milk, without sodium azide, and freeze it at -20°C. (BSA is not as effective for protecting your antibodies from the freezing process as milk is, but antibodies in almost any solution can be frozen and reused.)The tradeoff here is repeated freeze-thaw cycles, which can denature the antibody. Yet, chances are you’ll run out of solution before anything noticeable happens. To save solution during the handling of antibodies, keep your diluted antibodies in a container in which the blots are incubated.
Diminishing signal
Preserving good signal during detection is another concern. Most antibodies don’t suddenly stop working. In general, you’ll see only a slight difference in band intensity and blot quality obtained with freshly diluted and reused primary antibodies. Pay attention to how many times the antibody solution has been used and dispose of it when the signal begins to fade. For instance, anti-GFP antibodies can be reused up to 10 times, but a reduction in signal intensity may start to occur after four uses. It’s good practice to monitor the signal and prepare a new batch of the antibody as signal starts to weaken. In some cases, reusing an antibody has the added benefit of reduced background, because the limited amount of antibody shows increased specificity for the target with the highest affinity.
Product variation
How often you reuse an antibody also depends on the type of antibody and the manufacturer. Each antibody has a different affinity and stability. You may be able to use one type of antibody repeatedly over 15 to 20 days, but a different antibody over the course of just a week, regardless of storage conditions. Most cell-signaling antibodies, for instance, can be used three or four times within a span of 15 to 20 days. For some manufacturers, even if the antibody has been used just once, but is more than 20 days old, it’s worth throwing it out and starting with fresh product. Again, experimentation will help in this decision process.
Finally, very few researchers will reuse secondary antibodies, as you generally burn through fewer of them per equivalent volume. Secondary antibodies are also cheaper and used at far more dilute ranges. The decision to reuse antibodies overall is largely project dependent. In some experiments, the sample is more limiting than the reagents. If you’re saving $20 per Western by reusing an antibody, but risking a $100 sample, then it’s not efficient on a per-cost basis. Take a good look at your experimental design to determine where to best capture your savings—reusing antibodies is just one of many ideas.