I have been working in a lab in the Departments of Pediatrics and Human Genetics for almost 3 years. I wanted to stay to continue working over the summer and was looking for funding. I found the LSA Opportunity Hub and applied for an internship grant.
My lab studies chromodomain helicase DNA-binding protein 7, or CHD7. Loss-of-function mutations in CHD7 cause CHARGE syndrome, a rare (1 in 10,000 live births) developmental disorder. Since yesterday, I have been running a Western blot, which involves separating proteins by size and density, transferring them to a membrane, and using antibodies specific to that protein to determine how much protein is present in each sample.
The Western blot I am running right now contains samples from mouse embryonic stem cells (mESCs). One sample contains wild-type mESCs (Chd7+/+), one contains mESCs homozygous for the whirligig mutation, which is a nonsense mutation in Chd7 (Chd7 whi/whi), and one contains mESCs obtained from the U of M transgenic core. Our lab has had trouble detecting mouse Chd7 protein, so I am running this Western blot to determine whether the problem is with our antibody or with our cell lines.
The first step in a Western blot is to extract protein from samples. These samples are cells I have cultured and not directly from mice. Chd7 is a nuclear protein, so the both the cells and the nucleus must be lysed to obtain Chd7.
Once I extracted the protein, I prepared Western samples. The samples include the extracted protein, a reducing agent to break up the protein, and a colored buffer. They must also be boiled before use so that the protein is denatured.
After the samples were boiled and cooled, I ran the protein gel. This involves making the running buffer, setting up the Western apparatus (we have a box that holds the gel in place and connects to the power supply), and loading the samples into the wells. Each well is about a centimeter wide, but very narrow, so I have to use special gel-loading pipette tips to place the sample in the well.
The gel took about 1.5 hours to run. The next step is the transfer. After emptying the box of running buffer and setting up a nitrocellulose membrane, I cracked open the plastic casing of the gel and placed the gel on top of the membrane. The gel is only a few centimeters thick and has the consistency of firm Jell-O, so the transfer is the most difficult part of a Western blot. Once the gel and the membrane are in place, I laid them into the transfer cassette, encase between layers of blotting pads to ensure that the membrane and gel are tightly connected. During the transfer, the protein that was separated during the gel running is transferred onto the membrane, where it can be detected with antibodies
The transfer takes place in the cold room overnight. This morning, when the transfer was complete, I took out the membrane. I could see the clear bands of my ladder on the membrane, so I knew the transfer worked well. I washed the membrane in TBST (a buffer), then put it in 5% milk to block. Blocking involves submerging the gel in a protein solution (in this case, milk) and allowing the protein to bind to the empty spaces on the membrane, where the proteins in the sample are not located. This ensures that the antibodies will bind specifically to the protein I am interested in.
Over the weekend, I will block the proteins with the primary antibodies (there are two: one for Chd7, and one for a loading control that should be present in all samples). I have attached a picture of my bench. On the far left is the rotating block, where I washed the membrane and blocked it.