For our studies, quantification is the key. We find it easy to quantify blots on the Odyssey® Sa system.
Dr. Wendy Bollag
Georgia Regents University
By tapping into biochemical signal pathways, Dr. Wendy Bollag is interpreting how cellular responses are elicited. Bollag, Professor at Georgia Regents University and Research Career Scientist at the Charlie Norwood VA Medical Center, is specifically interested in decoding lipid signaling mechanisms involved in keratinocyte differentiation and aldosterone secretion.
Her research team has been awarded a VA Merit grant to study the effects of ultraviolet (UV) light exposure on the skin. "We have found that UV light activates protein kinase D, a signaling enzyme, and has a pro-proliferative, or pro-growth effect on epidermal keratinocytes1," says Bollag. "In addition, our group was the first to propose that aquaporin 3 cooperates with the lipid metabolizing enzyme phospholipase D, and forms a new lipid signal that regulates early differentiation of keratinocytes2." They are also investigating the elevation of very low-density lipoprotein (VLDL), and a corresponding stimulation in aldosterone production involving phospholipase D, in obesity3.
Robust System for Quantitative Westerns
The [Sa] imager is quantitative, and you get more of a linear response. We hardly ever use the darkroom now.
Monitoring protein activation and post-translational modification demands extensive analysis of protein expression by immunoblotting. Bollag’s lab owns an Odyssey® Sa Imager that is used for quantitative Western blotting. "For our studies, quantification is the key. We find it easy to quantify blots on the Odyssey Sa system. It seems to be sensitive and works well for most of our applications," she says.
In addition to ease of quantification, linearity, and reproducibility of data are equally essential to Bollag’s group. "Film doesn’t have very good linear range and enhanced chemiluminescence (ECL) or chemifluorescence (ECF) are, by their nature, not linear. If you use both film and ECL, then you’ve got double non-linearity." Since the lab switched to the Odyssey Sa imager, their darkroom usage has considerably dwindled. "The [Sa] imager is quantitative, and you get more of a linear response. We hardly ever use the darkroom now."
Reliable and Reproducible Protein Expression Analysis
We can get reproducible data [for three separate experiments] with the Odyssey Sa Imager, and that is another reason why I like it.
Within the biomedical research community, there are growing concerns regarding the reproducibility of published data. "Not everything always reproduces, just because we have got a lot of variables including the cells themselves," Bollag says. Hence, she requires her lab members to repeat all studies at least three times with three different cell preps, or more than three animals, and submit data for publication only when they have similar results. "We can get reproducible data [for three separate experiments] with the Odyssey Sa Imager, and that is another reason why I like it."
As a reviewer, Bollag expects that data submitted for publication have discernible details of changes in protein expression, validated with Western blots. "Many scientists rely on quantitative RT-PCR and assume that’s the be-all and end-all. While I really like qRT-PCR and we do a lot of it, it is the protein that does the job," she says. In one of their studies, they found by qRT-PCR that CYP11B2 mRNA expression, responsible for aldosterone production, went up with Pioglitazone treatment. But CYP11B2 protein levels were not elevated and aldosterone production was actually inhibited4. "So it is important to show that the protein has something similar going on as the mRNA." On a related note, she states that studies showing % inhibition or fold changes in protein expression should be accompanied by reproducible and quantifiable data from at least three separate experiments.
Like Bollag, researchers studying signaling mechanisms can obtain quantifiable protein expression data using the Odyssey Infrared technology.
- Vivek Choudhary, Lawrence O. Olala, Ismail Kaddour-Djebbar, Inas Helwa, Wendy B. Bollag; Protein Kinase D1 Deficiency Promotes Differentiation in Epidermal Keratinocytes; Journal of Dermatological Science; Available online 2 October 2014
- Vivek Choudhary, Lawrence O Olala, Haixia Qin, Inas Helwa, Zhi-qiang Pan,Ying-Ying Tsai, Michael A Frohman, Ismail Kaddour-Djebbar and Wendy B Bollag; Aquaporin-3 Re-Expression Induces Differentiation in a Phospholipase D2-Dependent Manner in Aquaporin-3-Knockout Mouse Keratinocytes; Journal of Investigative Dermatology (18 September 2014); doi:10.1038/jid.2014.412
- Ying-Ying Tsai, William E. Rainey, Zhi-qiang Pan, Michael A. Frohman, Vivek Choudhary, and Wendy B. Bollag; Phospholipase D Activity Underlies Very-Low-Density Lipoprotein (VLDL)-induced Aldosterone Production in Adrenal Glomerulosa Cells; Published Online: June 23, 2014; http://dx.doi.org/10.1210/en.2014-1159
- Zhi-qiang Pan, Ding Xie, Vivek Choudhary, Mutsa Seremwe, Ying-Ying Tsai, Lawrence Olala, Xunsheng Chen, Wendy B. Bollag; The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells; Molecular and Cellular Endocrinology; 25 August 2014, Volume 394, Issues 1–2, Pages 119–128.
- Dr. Geir Bjørkøy
University College of Sør-Trøndelag and CEMIR Centre of Excellence, NTNU
- Dr. Wendy Bollag
Georgia Regents University, Charlie Norwood VA Medical Center
- Dr. Jeremy Chambers
Florida International University, Miami, FL
- Dr. Paul Digard
University of Cambridge, UK
- Dr. Jane Gray
Cancer Research Institute, Cambridge, UK
- Manuela Gustafsson
Karolinska Institute's Department of Laboratory Medicine
- Dr. Oliver Hantschel
- Dr. Stefan Hüttelmaier
Martin Luther University
- Dr. Jelena Janjic
- Dr. Andrea Kasinski
- Dr. Sue Katz
Rogers State University
- Dr. Lisa Keyes
University of Florida College of Medicine
- Dr. Stanislav Kholmanskikh
Weill Cornell Medical College
- Dr. Patrick Kiely
University College Cork, Ireland
- Dr. Andreas von Knethen
Johann Wolfgang Goethe-University
- Dr. Michael Lee
University of Massachusetts Medical School
- Dr. Robert Lewis
University of Nebraska
- Dr. H. Charles Manning
- Dr. Debra Martin
Saint Mary's University of Minnesota
- Dr. Kevin Morano
The University of Texas Graduate School of Biomedical Sciences at Houston
- Dr. Rick Page
Miami University in Oxford, OH
- Dr. Nicolai Peschel
Universität Würzburg Department of Neurobiology & Genetics
- Dr. Wendy Picking
Oklahoma State University
- Dr. Martin Pomper
Johns Hopkins Medical School
- Dr. Vikash Reebye
Imperial College London, UK
- Dr. Rosemary Schuh
University of Maryland School of Medicine