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If you calculate the cost of the chemistry, recycling of film for silver extraction, service contracts and repairs, it is much more cost-efficient to use imaging systems.

Dr. Stanislav Kholmanskikh
Neurogenetics
Weill Cornell Medical College

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At the Brain and Mind Research Institute (BMRI) of Weill Cornell Medical College in New York, Dr. Stanislav Kholmanskikh and colleagues are paving the way for researchers to adopt 'sustainable' Western blotting practices.

Dr. Stanislav Kholmanskikh

The Assistant Research Professor and the Neurogenetics group of the BMRI decipher the effects of mutations in certain genes associated with nervous system development and regulation of neuronal cytoskeleton. "We want to know exactly how particular mutations in genes translate into abnormal proteins and how then those result in disease," explains Dr. Kholmanskikh.

His research group is studying gene mutations linked to the development of neural tube defects, neuronal migration disorders and conditions associated with impaired neural cell proliferation. They have studied1,2 mutations in LRP6 and CCND2 genes that generate mutant proteins, disrupting their respective cell signaling pathways; Wnt and (PI3K)-AKT, during embryonic development. "Our lab has identified a mutation in the LRP6 gene as the cause of folate-preventable neural tube defects in a mouse model," affirms Dr. Kholmanskikh. In other studies1, the group has shown that CCND2 has a significant developmental role in regulating brain size. The lab relies heavily on data from Western blotting experiments to provide answers to questions concerning protein detection and expression.

Closing The Door On Darkrooms

Dr. Kholmanskikh's group and other labs in his department at the BMRI have decisively abandoned darkrooms. "We do not have darkrooms in our new facility. We are definitely moving away from them, and it makes sense from an environmental and cost-efficiency standpoint" he insists. The Odyssey® Fc had a part to play in realizing this decision.

Dr. Kholmanskhikh wanted to contribute to reducing hazardous waste and protectingthe environment. Concerned with the hazards and waste generated by developing blots using film, as well as the inconvenience of sharing the facility with many other labs, his lab invested in an Odyssey Fc Imaging System. Shortly thereafter, he came to realize the benefits and conveniences that the technology offered.

An Enabling Technology

When using fluorescence detection on the Odyssey Fc, I can evaluate a loading control and my protein of interest (Lis1) all in the same blot. This has been tremendously advantageous for the lab.

"The quantification aspect is very important for us," states Dr. Kholmanskikh. Prior to the Odyssey Fc, when the lab had been using chemiluminescence detection, they would perform 12 serial dilutions of each sample. They would then look for linear changes in signal intensity between these dilutions so as to estimate quantitative differences between them. "When the lab switched to Odyssey Fc and started doing fluorescence detection, initially I did those 12 dilutions and found that actually every single one of them was in linear range, as opposed to usually 3 out of 12 for chemiluminescence," he explains. Their lab has converted entirely to fluorescence detection and doesn't feel the need to do serial dilutions anymore.

The Odyssey Fc has enabled their lab to detect phosphorylated vs. total protein on the same blot. One of the group's recent publications presents total and unphosphorylated (active form) β-Catenin probed together using the instrument2. In addition, the Odyssey Fc has also facilitated the expression analysis of similarly-sized proteins. Lis1, one of the proteins being studied by the lab, and the loading controls used for experiments have comparable molecular masses. Dr. Kholmanskikh says, "With chemiluminescence, I would have to strip and re-probe with another antibody to provide a loading control. When using fluorescence detection on the Odyssey Fc, I can evaluate a loading control and my protein of interest (Lis1) all in the same blot. This has been tremendously advantageous for the lab."

Sustainable Western Blotting

Dr. Kholmanskikh asserts, "One has to realize that the cost of film may be cheaper for one experiment — but that's only for one experiment. If you calculate the cost of the chemistry, recycling of film for silver extraction, service contracts and repairs, it is much more cost-efficient to use imaging systems."

In addition to the Odyssey Fc, Dr. Kholmanskikh has access to an Odyssey CLx Imaging System that was purchased by the department, upon his recommendation. He is excited to advance his research through the multiple applications that the Odyssey CLx supports.

Environmentally-conscious researchers like Dr. Kholmanskikh are taking advantage of a solid technology platform while reducing their ecological footprint with the Odyssey Imaging Systems.

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Papers From Kholmanskikh Lab Citing Odyssey Fc

  1. Mirzaa GM, Parry DA, Fry AE, Giamanco KA, Schwartzentruber J, Vanstone M, Logan CV, Roberts N, Johnson CA, Singh S, Kholmanskikh SS, Adams C, Hodge RD, Hevner RF, Bonthron DT, Braun KP, Faivre L, Rivière JB, St-Onge J, Gripp KW, Mancini GM, Pang K, Sweeney E, van Esch H, Verbeek N, Wieczorek D, Steinraths M, Majewski J; FORGE Canada Consortium, Boycott KM, Pilz DT, Ross ME, Dobyns WB, Sheridan EG.
    De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.
    Nature genetics, 2014 May;46(5):510-5. doi: 10.1038/ng.2948. Epub 2014 Apr 6.
  2. Gray JD, Kholmanskikh S, Castaldo BS, Hansler A, Chung H, Klotz B, Singh S, Brown AM, Ross ME
    LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.
    Human molecular genetics, 2013, Vol. 22, No. 21, doi: 10.1093/hmg/ddt277. Advance Access published on June 16, 2013. 4267-4281. 2013.


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