Increasing Western Blot Data Output
The Western Blot is one of the first and most commonly utilized tools that researchers go to during the discovery process. However, once your target has been confirmed and your antibodies validated there is very little reason to stick with the Western Blot due to its technically challenging nature and inability to increase throughput. At this point, researchers often turn to higher throughput methods such as ELISA.
What if we didn’t cast this tried-and-true method aside when the time comes to scale up data output? I know what you’re thinking, how can I possibly scale up my data output when I only have 12 lanes on my gel? It’s simple, skip all the steps that make Western Blotting a time consuming and technically challenging hassle. Yes, that means keep it simple and use a dot blot! At this point you have a validated antibody that you know is specific for your target, so there is no reason why you can’t make the switch to increase your throughput. Using a dot blot will decrease your assay cost and allow you to run multiple replicates of your samples and controls all without the variability caused by gel electrophoresis and electrotransfer. Let’s be honest here, an ELISA is just a glorified dot blot for which you have to search for a matched antibody pair. Screening antibodies, optimizing an ELISA and buying a plate reader are all expenses that could easily be avoided if we just simplify our blotting process with dots.
Increasing your data output will finally give you the power to truly call your blots quantitative. An added benefit to using a dot blot is that the increased surface area of your sample will enable you to increase the linearity and dynamic range of your Western blot. When detecting a low abundance target it is not uncommon to increase your sample load. We often forget that even though we load more sample, it doesn’t necessarily improve sensitivity since the membrane has a limited binding capacity. Lysates are complex mixes of proteins that are broadly distributed. It is very likely that the sliver of real estate that your target occupies is approaching the membrane’s binding capacity at high protein loads, making it difficult to detect.
Figure 1: Serially diluted HeLa Lysate (N=6) probed with a mouse anti-GAPDH primary antibody followed by a goat anti-mouse HRP conjugated secondary detected with WesternBright™-ECL Substrate.
When you’re ready to simplify your workflow, Advansta has you covered with substrates that respond linearly over a broad dynamic range. Both WesternBright™-ECL and WesternBright™-Quantum are well suited for increasing your linear dynamic range with dot blotting.
Advanblock™-Chemi Blocking Solution
HRP Conjugated Secondary Antibodies
WesternBright™ ECL Substrate
WesternBright™ Quantum™ Substrate