Minute™ Adipose Tissue Fractionation Kit (20 Preps)

Manual & Protocol | MSDS

On average, total cellular proteins account for less than 2% of adipose tissue. Fractionation of adipose tissue is technically very challenging due to its high lipid and low protein contents. Adipose tissue, especially white adipose tissue (WAT), has been recognized as an important endocrine and inflammatory organ in addition to its energy storage function. Fractionation and analysis of proteins from adipose tissues are critical for understanding many physiological/pathological conditions. The water-oil emulsion present in biological samples is notoriously difficult to separate. We have developed a novel technology to deal with this issue. A porous filter with unique surface properties and pre-defined pore size, coupled with a specially formulated detergent-free fractionation buffer, is the key to fractionating adipose tissue into two fractions: a water-soluble protein fraction containing mainly cytosolic proteins and a water-insoluble fraction containing mainly plasma membrane and organelles such as mitochondria. The buffers used in this kit are free of primary amines, detergents, and reducing agents. Isolated proteins are compatible with all downstream applications, including TMT labeling, enzyme digestion, MS analysis, and other applications.

A. SDS-PAGE (10%) profiles of fractionated adipose tissues. Lane 1, a water-soluble fraction of porcine WAT; Lane 2, a water-insoluble fraction of porcine WAT; Lane 3, a water-soluble fraction of rat WAT; Lane 4, a water-insoluble fraction of rat WAT.

B. Western blottings of fractionated proteins from rat WAT; Lane 3. water-soluble fraction; Lane 4. Water-insoluble fraction. Proteins were separated in 10% SDS-PAGE and probed with the following cellular protein marker antibodies: Anti-Na/K ATPase alpha1, a plasma membrane marker (Upstate, clone 464.6), anti-lamin B1, a nuclear envelope marker (ab16048, Abcam Cambridge, MA), anti-ubiquinol-cytochrome C reductase core protein (Abcam, ab 96333) and GAPDH, a cytosolic marker (Sigma). The specific protein bands were visualized by a substrate Opti-4CN (Bio-RAD). 

Kit includes:

References (3)

1. Markan, K. R., Naber, M. C., Small, S. M., Peltekian, L., Kessler, R. L., & Potthoff, M. J. (2017). FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue. Molecular Metabolism. http://dx.doi.org/10.1016/j.molmet.2017.03.009
2. Liu, X., Tong, W., Zhao, X., Zhang, H., Tang, Y., & Deng, X. Chinese herb extract improves liver steatosis by promoting the expression of high molecular weight adiponectin in NAFLD rats. Molecular Medicine Reports.
3. Subbaramaiah, K., Iyengar, N. M., Morrow, M., Elemento, O., Zhou, X. K., & Dannenberg, A. J. (2018). Prostaglandin E2down-regulates sirtuin 1 (SIRT1) leading to elevated levels of aromatase, providing insights into the obesity-breast cancer connection. Journal of Biological Chemistry, jbc-RA118.