How to Make a Good Western Blot

Western blotting is a great way to find out more about proteins. Western blotting is a common method used by everyone from first-year students to seasoned researchers. It has a number of uses. In general, it is useful for identifying, semi-quantifying, and figuring out the size of specific proteins.

Immunoblotting and primary antibody western blot are other names for Western blotting because it uses an antibody to find a specific antigen. The process of moving the proteins from a gel to a membrane is called "blotting. The main point of western blotting is to use gel electrophoresis to separate proteins from a sample that has a mix of native and denatured proteins. After that, the proteins are put on a membrane. By washing the membrane in a solution that contains the primary antibody, which should bind to the protein of interest, the target protein is marked.

But, as with any lab process, there are a lot of things that could go wrong! Here are a few important things to know about the western blot.

How should I get the samples ready?

If you want to use a sample of tissue, you will first need to break it up by homogenizing it. Add a buffer so that the cells can be broken down and the proteins can be made to dissolve. To keep proteins from breaking down, protease and phosphatase inhibitors can be added to the buffer.

Most of the time, but not always, Western blotting is done with proteins that are denaturated. This makes sure that proteins are separated by size during electrophoresis. Most samples can have their proteins denatured by boiling them for five minutes and using denaturing condition during electrtrophoresis.

What kind of gel do I need?

Polyacrylamide (PA) gels and a sodium dodecyl sulfate (SDS) buffer are used in most western blots. Most Western blots that use denatured proteins use an electrophoresis method called SDS-PAGE. During electrophoresis, SDS makes all of the proteins negatively charged. This lets the proteins be separated by their molecular weight. The quality and amount of protein transferred to membrane can be affected by the thickness of the gel. Most of the time, thinner gels allow for more efficient protein transfer, which leads to more clearly defined protein bands.

Should I use a blocking agent?

When electrophoresis is done, the proteins are put on a membrane so that antibodies can bind to them. Then, a blocking agent is used to stop the primary antibody from binding to the membrane increasing background. Blocking agents are often made of non-fat dry milk or bovine serum albumin, along with a small amount of detergent. The proteins in the blocking solution should bind to the membrane in places where the target protein has not yet bound. This keeps the primary antibody from binding to the membrane, which should reduce the "noise" in the background and make the results clearer.

How do I figure out how much primary antibody to use?

Try to use a concentration of antibody that gives the best balance between signal and background. A solution with an antibody concentration of 0.5g/mL to 5g/mL could work, but most of the time a concentration of 1g/mL is enough. You should also think about how common the target protein is. If you want to find low-abundance proteins, you might need to try increasing both the amount of protein and the concentration of the primary antibody.

How can I make the time in the incubator work best?

The standard way to see the target protein is to let the primary antibody mix with the sample for one hour at room temperature. But an overnight incubation at 4°C should give the antibody-antigen reaction plenty of time, making it more likely that a positive signal will be shown. It also help to perform all washing steps at 4°C, that can be done using BlotCycle, automated western blot processor.

How do I use the controls?

To make sure your western blotting results are correct, it is important to use both positive and negative controls. You can use the secondary antibody by itself as a negative control. This will help you figure out how much of the background is due to the secondary antibody. Don't forget St. John's Antibody Validation Project when you need to find antibodies for Western blotting. As part of the Project, you can get up to five free samples of our primary antibodies in trial sizes. There are about 12,000 antibodies in the project, so we're sure you'll find something that works for you.