West side story

About two months back, I’d promised a post about western blots. So here goes.

The principle behind a western blot is the same as electrophoresis. Proteins acquire a negative charge in an appropriate solution. So when a potential gradient is applied, protein molecules will naturally move to the positive electrode.

However, not all proteins are of the same size. So if we place some kind of size-based filter between the electrodes, the smaller molecules would move to the electrode faster. That is the function of the gel in gel electrophoresis. There are pores of a certain size in the gel (the size of the pores can be varied based on the ingredients used) So under a certain potential gradient, with a gel of a certain pore size a protein of certain size/mass can only travel a certain distance in a certain period of time. So a mixture of proteins can be separated on the basis of size by gel electrophoresis. First we stain the proteins before we add them to the gel. That way, we can see where the proteins have been added (A protein solution in water is usually colourless) Alongside the proteins that we study, we also need to add markers. Markers are proteins of a predefined size, which we use like a ruler to gauge the size of the protein we’re studying.

Now that we’ve separated out the mixture of proteins, we need to make sure that the protein we’re looking for is in there. That means detecting with antibodies, since antibodies are extremely specific with the proteins that they bind to. But antibody staining can’t be done in a gel. So the proteins need to be transferred to a durable film of nitrocellulose or polyvinylidene fluoride (PVDF). Once the transfer is complete, antibody staining is done. Staining is a two step process. First, we stain with an antibody that’s specific for the protein we’re interested in. Next we stain with another antibody that’s both specific to the first antibody, and also has fluorescence activity

This is important because it simplifies the procedure. To induce fluorescence activity in an antibody, it needs to be modified artificially. By separating protein recognition from fluorescence, we only need to produce one kind of secondary antibody, which will bind to all primary antibodies regardless of what protein the primary antibody binds to. Instead of building fluorescence into primary antibodies, which means tinkering with pretty much every antibody produced for western blotting.

Why is fluorescence important? It helps us see precisely where our protein is located on the PVDF film and, as a result, on the gel (since the film is like a photocopy of the separation pattern on the gel)

Although I’ve simplified things here, the whole procedure takes a day at least to carry out. Electrophoresis can take anywhere up to 4 hours depending on the protein you’re studying. Transfer of protein onto a PVDF film is done overnight. Primary staining takes three hours, secondary staining takes an hour. And after each staining, the film needs to be rinsed for an hour, otherwise there’s a lot of background fluorescence and you can’t see your protein of interest clearly.

In other news, I’ve shifted lab groups for this semester. My previous guide is on maternity leave till April, so work in her group will go slowly till then. This semester, I’ll still be working on cell cultures. But this time it’ll be fly cell lines, not human. These cells are generally hardier than both HeLa and 293T, so I should have an easier time, in terms of cells dying, over the next few months. Wish me luck!