Children’s Hospital Oakland Research Institute (CHORI) is using Bio-Rad’s TGX Stain-Free gels to demonstrate how target genes regulate low-density lipoprotein receptor (LDLR) protein.
Dr. Frederick Bauzon, a former postdoctoral researcher in Dr. Ronald Krauss’s lab at CHORI who now serves as a professor at California State University, East Bay, investigated the role of a candidate gene associated with HMGCR transcript levels discovered in a linkage study.
The study found that reducing LDLR expression decreases the amount of available receptor protein, which normally binds LDLs circulating in the bloodstream and transports them back inside the cell.
The pathway was discovered 20 years ago and has been of interest in drug target development due to its regulating role.
“We were trying to show how this target gene regulates LDLR. Does it regulate the protein directly, or is LDLR regulated at the mRNA level? We investigated mRNA levels through qPCR, but ultimately, we want to demonstrate that [the level of] the gene is also reduced using SDS-PAGE and western blotting,” says Bauzon. “Stain-free gels save us considerable time in that portion of our research.”
Previously when running western blots in graduate school, Bauzon used regular Tris-HCl gels and found that gel staining and destaining was enormously time-consuming.
It typically took two–three days to complete a western blot. He welcomed the switch to Bio-Rad’s TGX Stain-Free precast gels for their experiments in the Krauss lab.
The gels feature a proprietary chemistry that eliminates the need for staining, providing protein separation and imaging capabilities in approximately 25 minutes.The convenience of using Bio-Rad’s stain-free precast gels eliminates Coomassie and Ponceau S staining, saving researchers valuable time.
“Back in grad school, I would hand cast everything but gradient gels,” says Bauzon. “It took two hours to run a single gel. Using the Bio-Rad gel system I ran gels in a half hour, and this means getting more research done.”
Bauzon noted that with a limited number of power supplies, gel boxes, and other equipment, as well as a premium on bench space in the lab, it’s most efficient to run multiple gels in one tank using the same buffer.
The Krauss lab used the 12% Mini-PROTEA TGX Stain-Free precast gels to run the LDL proteins (~115–120 kD). The western blots are subsequently developed using chemiluminescence to visualise the proteins.
When he started his postdoctoral research at CHORI, Bauzon worked with the bioinformatics specialists in the lab and functionalised suitable genes based on their expression levels with HMGCR transcript levels.
He then ran experiments in triplicate (without quantitation) for roughly one year to demonstrate a clear reduction in LDLR upon knockdown of the candidate gene. All the experiments demonstrated a reduction in LDLR and the protein levels after the gene was silenced.
“The use of gels was obviously just one component of our research, but little things like having premarked lanes and not having to use a Sharpie to demarcate lanes that can smear off makes a difference,” says Bauzon. “A lab will always work with what is easiest and most comfortable to use.”
The overall objective in the Krauss lab was to identify mutations involved in cholesterol homeostasis, specifically through two research projects:
- Investigating the genetic and metabolic determinants of atherogenic dyslipidemia — high triglyceride, low HDL, and increased LDL levels — associated with obesity and insulin resistance in humans
- Identifying genetic influences in response to dietary and drug treatment of atherogenic dyslipidemia. Bauzon hopes that the research will ultimately support this mission by providing future drug targets for the therapeutic control of LDL uptake.
