Posts Tagged ‘tools’

Designing the Perfect Quantitative Western Blot

 :: Posted by American Biotechnologist on 04-02-2014

A methods article published yesterday provides a rigorous and concise workflow with specific instructions on how to produce and analyze quantitative data using western blot experiments. The paper, coauthored by Bio-Rad scientists and published in BioMed Research International, also highlights recently introduced technologies that improve reproducibility. The result is a powerful, step-by-step guide to obtaining quantitative and reproducible densitometric data from western blots regardless of the specific experiment.

Although western blotting is a well-established laboratory technique, it has recently come under fire as a quantitative method because extreme care must be taken when generating and interpreting the resulting data.

The technique is challenging and requires following a rigorous methodology to achieve reproducible and quantitative data. According to a recent survey of more than 750 labs, 41% of researchers say their western blots fail a quarter of the time.

Dr. Aldrin Gomes, an assistant professor at University of California, Davis, agrees that flawed western blots are not unusual. To compare expression of a protein of interest from sample to sample, protein abundance is commonly normalized to a housekeeping gene. “When I see a large, dense band for the protein of interest or the housekeeping protein, I cringe,” says Gomes. That dense band usually means the protein of interest or housekeeping protein was no longer within the assay’s linear dynamic range. No accurate quantitative data can be extracted from such blots.

Sean Taylor discusses the BioMed Research International paper he coauthored.

Another common reason for failure of quantitative western blots is flawed or incomplete protocols, according to Sean Taylor, the paper’s lead author and a Bio-Rad field application scientist (watch a video of him discussing the paper on the left). To address this, Taylor’s review pays special attention to experimental design and sample preparation and discusses proper definition of the linear dynamic range of protein loading, all key factors for generating meaningful quantitative western blot data.

Taylor also introduces more advanced concepts to improve reproducibility, simplify workflow, and reduce the time and cost of western blotting. One such technique is stain-free total protein normalization, which over the past year has proven superior to using housekeeping proteins or total protein staining to correct for loading errors.

With this article, Taylor hopes researchers now have a simple guide to ensure quantitative and reproducible western blot data for all research fields that rely on this technique.

To read the open access research article, visit http://bit.ly/1kCAOcr.

For additional resources please consult Bio-Rad’s guide to Troubleshooting Western Blots.

Video: How to Perform Perfect Quantitative Western Blotting

 :: Posted by American Biotechnologist on 04-01-2014

Lab on a chip….on a cell phone!

 :: Posted by American Biotechnologist on 11-18-2013

In developing nations, rural areas, and even one’s own home, limited access to expensive equipment and trained medical professionals can impede the diagnosis and treatment of disease. Many qualitative tests that provide a simple “yes” or “no” answer (like an at-home pregnancy test) have been optimized for use in these resource-limited settings. But few quantitative tests—those able to measure the precise concentration of biomolecules, not just their presence or absence—can be done outside of a laboratory or clinical setting. By leveraging their discovery of the robustness of “digital,” or single-molecule quantitative assays, researchers at the California Institute of Technology (Caltech) have demonstrated a method for using a lab-on-a-chip device and a cell phone to determine a concentration of molecules, such as HIV RNA molecules, in a sample. This digital approach can consistently provide accurate quantitative information despite changes in timing, temperature, and lighting conditions, a capability not previously possible using traditional measurements.

Read more…

Extracting and Purifying Human DNA in Under Three Minutes

 :: Posted by American Biotechnologist on 05-07-2013

University of Washington engineers and NanoFacture, a Bellevue, Wash., company, have created a device that can extract human DNA from fluid samples in a simpler, more efficient and environmentally friendly way than conventional methods.

Conventional methods use a centrifuge to spin and separate DNA molecules or strain them from a fluid sample with a micro-filter, but these processes take 20 to 30 minutes to complete and can require excessive toxic chemicals.

UW engineers designed microscopic probes that dip into a fluid sample – saliva, sputum or blood – and apply an electric field within the liquid. That draws particles to concentrate around the surface of the tiny probe. Larger particles hit the tip and swerve away, but DNA-sized molecules stick to the probe and are trapped on the surface. It takes two or three minutes to separate and purify DNA using this technology.

Read the full story on the UW website.

How an iPhone a day keeps the doctor away

 :: Posted by American Biotechnologist on 10-04-2011

In a feat of technology tweaking that would rival MacGyver, a team of researchers from the University of California, Davis has transformed everyday iPhones into medical-quality imaging and chemical detection devices. With materials that cost about as much as a typical app, the decked-out smartphones are able to use their heightened senses to perform detailed microscopy and spectroscopy. The team will present their findings at the Optical Society’s (OSA) Annual Meeting, Frontiers in Optics (FiO) 2011, taking place in San Jose, Calif. Oct. 16-20.

The enhanced iPhones could help doctors and nurses diagnose blood diseases in developing nations where many hospitals and rural clinics have limited or no access to laboratory equipment. In addition to bringing new sensing capabilities where they are needed most, the modified phones are also able transmit the real-time data to colleagues around the globe for further analysis and diagnosis.

“Field workers could put a blood sample on a slide, take a picture, and send it to specialists to analyze,” says Sebastian Wachsmann-Hogiu, a physicist with UC Davis’ Department of Pathology and Laboratory Medicine and the Center for Biophotonics, Science and Technology, and lead author of the research to be presented at FiO.

Read more from the Optical Society…