Protein transfer from the gel to membrane is the most variable part of a Western blot. Even if you know the optimal buffer and transfer conditions, there are always bubbles, uneven protein transfer or other transfer artefacts, which you don’t know about. And they usually occur in the most prominent place on the membrane.
So how do we solve the problem of seeing invisible proteins on a white membrane?
Blue: Using Coomassie to detect transfer
A first thought would be to stain the gel after transfer. After all, you use the prestained protein markers to check if the transfer works at all. Why not just stain the whole gel? Hint: If it’s that obvious but nobody routinely does it, there’s a catch.
The catch is that, while staining the gel with Coomassie before transfer sort of works, the process is inefficient. The classic Coomassie stain consists of incubating the gel in a mix of methanol and acetic acid, which works as a solvent for the stain. But alcohol and acid treatment is harsh. It fixes the protein inside the gel, interfering with the transfer. If you pre-stain your gel, you will leave a lot of protein behind.
If you accidentally stained a gel with the last of your samples, as a last-ditch effort, you can still use the gel for transfer. Just use a nitrocellulose membrane, and increase your normal transfer and exposure time three-fold. And be prepared to lose about 75% of your proteins.
Of course, you can also try commercial preps of protein-staining solutions, which are called Colloidal Coomassie Stains (for example this stain from BioRad). Colloidal Coomassies usually have a better sensitivity and require less harsh treatment than homemade Coomassie stain. But I have an even better idea for you.
Red is the new blue: Ponceau S for membrane staining
Imagine that you can stain not a pre- or post-transfer gel, but the membrane itself, be it a PVDF or nitrocellulose membrane, and that in five minutes, you can see your proteins on the membrane in all their glory. Admittedly, you can also discover a large artifact bang in the middle of your membrane. This will annoy you but will prevent wasting reagents. And more importantly, this will save your time squandered ongoing through the rest of the Western dance motions with no image at the end to put in your groundbreaking article.
Good news: We have a dye that does all that and more – Ponceau S aka Acid Red. The dye is non-toxic, and one of its derivatives used in Europe as a food dye. If the colour of Coomassie, as it says on the jar, is “brilliant blue,” although it has a different chemical structure, Ponceau can be described as “brilliant red.” A water-based solution of Ponceau S stains the protein bands on the membrane in a pinkish-red colour.
As with Coomassie, there is some background, but you can easily destain the membrane with water. Changing the solution several times reduces background; prolonged incubation destains the bands as well, returning the membrane to its white hue. But, you don’t need to de-stain the bands on the membrane completely after dying with Ponceau S. The dye will come off during your block equilibration. You also don’t need to mind your primary antibodies solution turning pink as well: traces of Ponceau S do not interfere with the antibodies’ binding.
But, Ponceau S benefits don’t stop after your Western detection. The proteins are still on the membrane. If you didn’t take a picture of your Ponceau-stained membrane for your lab book before, you can stain the membrane after you are done with it. Dry it out and keep it for future loading reference. Like a good mascara, dry Ponceau staining doesn’t smudge.
Frankly, given that staining with Ponceau is so fast, efficient, and devoid of negative effects, it’s surprising that people don’t use it more often. You can give it a try using our AdvanStain Ponceau.
Photo courtesy of Doug Kerr