Reinventing the Western Blot?

August 15, 2014 in Blog by jmeyer

By Dr. Joanne Elliott

Western Blotting has been a common technique in molecular biology laboratories for more than 30 years. This mainstay technique uses gel electrophoresis to separate native or denatured proteins in a tissue homogenate or protein extract. These proteins are then transferred onto a nitrocellulose or PDVF membrane, which can then be stained in a solution containing antibodies directed against the protein of interest. After staining with the primary antibody, the membrane is washed and incubated in a solution containing a secondary antibody linked to a reporter to enable detection. During detection the reporter (such as HRP) can cleave a chemiluminescent agent to produce luminescence. The amount of luminescence produced reflects the abundance of protein when normalized to a protein standard that should remain constant between samples.

The technique can provide researchers with information about a protein’s molecular weight, abundance, as well as expression pattern. Western Blotting can also be used to study post-translational modifications such as phosphorylation or protein-protein interactions. Today, Western Blotting is still the definitive diagnostic test to confirm HIV, BSE (Bovine Spongiform Encephalopathy), as well as some forms of Lyme disease, Hepatitis B and Herpes Simplex virus infection.

Three independent research groups in Seattle, Stanford and Brasil all deserve credit for developing the methods we use today to detect specific proteins using antibodies. However, apart from advances in Western Blotting detection methods and other small tweaks, the common principle of the Western Blot has remained unaltered since publication in 1979. Although Western Blotting is a tried and trusted technique, the method has many disadvantages. The entire procedure is very time consuming and manually cumbersome. Moreover, Western Blotting requires micrograms of protein and the number of samples that can be analyzed is often defined by the size of the gel. In an attempt to address these problems, researchers at UC Berkeley led by Amy Herr, have tried to reinvent the Western Blot. They recently published their work entitled “Single Cell Western Blotting” in Nature Methods in June 2014. The group developed a microscale Western Blot that can be used to analyze approximately 2000 individual cells in a microscope slide format. The technique starts by applying a suspension of single cells onto the polyacrylamide gel containing microwells. Once excess cells are washed away, those that have settled into the wells are rapidly lysed and solubilized within 10 seconds. An electric field is then applied across the slide to drive the proteins through the walls of the microwell. Proteins are separated within 500μm before UV light is used to covalently attach the proteins to the gel. Immunostaining with a primary and secondary antibody can then occur before the protein of interest is analyzed using a standard fluorescent microarray scanner.

This new method certainly has some advantages compared to the traditional Western Blot. Since individual cells can be studied, issues of heterogeneity in protein extracts are eliminated. In addition, the technique requires only 0.01 to 0.5 micrograms of protein which means protein characterization within rare cell types is achievable. The rapid cell lysis and protein separation also eliminates the laborious time consuming steps of a traditional Western Blot. The inventors are currently collaborating with industrial partners to commercialize their single cell Western Blotting technique. However, time will tell if researchers are ready to adopt this new approach to protein analysis.

Initial Findings of the Human Proteome Project

June 16, 2014 in Blog by jmeyer

By Dr. Joanne Elliot

Thirteen years after the completion of the Human Genome Project, we now have the first drafts of the human proteome. The goals of the Human Proteome Project were to “…..characterize all 20,300 genes known in the genome, to generate a map of the protein based architecture of the human body and to become a useful resource to help elucidate biological and molecular function to advance diagnosis and treatment of disease”.

Thanks to the efforts of two independent research teams, we now have access to vast quantities of data that will hopefully revolutionize our understanding of the human proteome. One group, based in Germany (led by Professor Kuster), analyzed protein isolated from 60 human tissues, 13 body fluids and 147 cancer cell lines. The other, with research teams in Baltimore and Bangalore (led by Professor Pandey), isolated proteins from 30 healthy human tissues including 7 fetal tissues, 6 types of hematopoeitic cells and 17 adult tissues. Both groups used mass spectrometry to create a catalogue of proteins in the sample mix and similarly, both published their findings in the same journal of Nature on May 29th, 2014. For the benefit of the scientific community, both teams have made their results available online in searchable databases. Data from Prof Kuster’s group can be found freely at while results from the Prof. Pandey’s team is available at

Prof Kuster’s team successfully detected 92% of proteins predicted to be encoded by the gnome, while Prof Panday’s group confirmed 84%. Interestingly, the initial findings of the human proteome have identified 193 novel proteins. These newly identified proteins seem to be transcribed from sections of DNA that were previously characterized as non-coding. In contrast, researchers were unable to locate approximately 2000 proteins that should be present according to genome analysis. These results ultimately raises questions about our understanding of how genes are transcribed and translated. Likewise, although we now have a comprehensive list of proteins, we cannot fully appreciate the function of each protein without considering how protein function may be altered by post-translational modifications, structure and protein-protein interactions.

Looking ahead, it is hoped that the expression profile of each protein may give researchers clues to their function. For example, if expression of a particular protein is restricted to a certain cell type, this may highlight which proteins are involved in development or homeostasis. Moreover, the human proteome may also give us a remarkable insight into how protein expression may be altered in disease. This information may help our understanding of disease development and progression. In the long term, the expression profile of certain proteins may even be used to determine the sensitivity of cells to a particular therapy or predict possible side effects of treatment. This would ideally lead to a more targeted therapeutic regime or personalized treatments for each patient.

As well as being an incredible achievement, the availability of the data generated by the Human Proteome Project will certainly mold and direct current and future research into protein expression and function in health and disease.

Article Published on The OutsourcePharma Advisory Board

June 3, 2014 in Blog by jmeyer

A new article published on Outsourcing-Pharma highlights The OutsourcePharma Advisory Board and its comprehensive CRO Reviews database.

Welcome Blog Post

May 13, 2014 in Blog by jmeyer

Welcome to The OutsourcePharma Advisory Board and our blog.  We are glad you are here and registered as an advisory board member. I’d like to take this opportunity to formally welcome you and give a little more background about the site and what’s in store for the coming weeks….It’s really an exciting time!

After more than a decade of serving the CRO industry with voice-of-the-customer market research and strategic consulting services, Life Science Strategy Group decided it was time to take its panel of biopharmaceutical industry professionals to the next level. Our idea first started out with the establishment of an online community dedicated to biopharmaceutical outsourcing professionals (like you). The goal of the community being to provide a place where Biopharma drug development and outsourcing professionals could communicate, ask questions in forums, network and share resources with other like-minded professionals. The closed, online community would also serve as a centralized portal where members could access all of Life Science Strategy Group’s active market research studies.

Well, one good idea led to another and here we are today. In addition to the above elements, we have taken the concept to a whole ‘nother level. We have added in a repository for various resources (web links, articles, white papers, reports), created defined member groups and built in quick polls that explore current industry topics. But we did not stop there – we went ahead and created the first and only Yelp-Like directory of customer reviews of CROs, which give our members the ability to read, search AND create anonymous reviews of the CROs they work with. On top of this we went ahead and incorporated several of the CRO performance metrics that are part of the vendor selection process and indicative of quality service delivery. We are sure you will quickly see the value as you discovery the nuggets of insight they contain.

Looking ahead, we have more planned for the site including timely blog posts, quick-poll site surveys with real-time results for members, new CRO reviews, opportunities for guest authors on our blog, sponsored-content on the site by leading CROs, and of course, more opportunities to participate in LSSG studies. We are also open to ideas shared by our community for further enrichment.

So take some time and explore the site. As an online community the more content and insight you contribute the better our community will be. We are also offering small cash incentives/ credits for completing various activities within the community including participation in LSSG’s studies, submission of CRO reviews and referring colleagues to The OutsourcePharma Advisory Board. “Follow” us on Twitter @OutsourcePharma to stay on top of the latest community announcements, news and opportunities to participate.

Thank you again. We look forward to hearing from you and will “see” you on the site.

– Jon