Description In average, total cellular proteins account for less than 2% of adipose tissues. 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 inflammation 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 sample is notoriously difficult to separate. We have developed a novel technology to deal with this issue. A porous filter with unique surface property and pre-defined pore size coupling with a specially formulated detergent-free fractionation buffer is the key to fractionate adipose tissue into two fractions: water-soluble protein fraction containing mainly cytosolic proteins and water insoluble fraction containing mainly plasma membrane, and organelles such as mitochondria. The buffers used in this kit are free of primary amine, detergent and reducing agents. Isolated proteins are compatible with all downstream applications including TMT labeling, enzyme digestion, MS analysis and other applications.

Application MS analysis, 2D gel analysis, ELISA, SDS-PAGE, immunoblottings, immunoprecipitation, enzyme activity assays, and other applications.

Kit components

1. 15 ml buffer A

 2. 15 ml buffer B

3. 1.5 ml microfuge tube (20)

 4. Pestles for 1.5 ml tube (2)

5. 20 protein extraction filter cartridges with collection tubes

6. Protein Extraction Powder (2 g)

Storage: Store the kit at 4C
Additional Materials Required: Table-Top Microcentrifuge with a maximum speed of 14,000-16,000 rpm.
Important Product Information The use of protease inhibitors is optional. However if downstream application takes significant amounts of time or the protein extract will be stored for longer period of time, addition of protease inhibitor to buffer A is recommended. For determination of protein concentration BCA assay (Pierce) is recommended. For protein phosphorylation studies, cocktails of phosphatase inhibitors need to be added to buffer A prior to use. Perform
all centrifugation steps in a cold room or in a refrigerated microcentrifuge.