Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes

Adipose tissue, especially white adipose tissue (WAT), has been recognized as an important endocrine and inflammation organ in addition to its energy storage function. Isolation and analysis of proteins from adipose tissues are increasingly critical for understanding many physiological/pathological conditions. However, isolation and analysis of WAT and brown adipose tissue (BAT) are technically very challenging due to their high lipid and low protein contents. The water-oil emulsion present in biological sample is notoriously difficult to separate.

Invent Biotechnologies has developed a novel technology to deal with this issue. A porous filter with unique surface property and pre-defined pore size and thickness coupled with a specially formulated detergent-free extraction buffer is employed to rapidly and effectively separate water-oil emulsion derived from adipose tissue homogenate. The extraction buffer has lower freezing point than that of oil in adipose tissues, and the aqueous phase can be quickly separated from oil phase by passing the tissue homogenate through the filter. The total proteins isolated are unbiased representation of cellular proteins in the tissue. The extracted proteins concentration is very high (2-3 mg/ml) as compared to other methods.

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


Images of Water-Oil Emulsion of Porcine WAT Before (Left) and After (Right) Passing Through the Filter.


A. SDS-PAGE (10%) Profiles of Total Protein Extracted from Different Adipose Tissues.

Lane 1, porcine WAT; Lane 2, chicken BAT; Lane 3, rat WAT.
 

B. Western Blottings of Extracted Proteins from Rat WAT.

Proteins were separated in 8-16% gradient SDS-PAGE and probed with 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 components                                                                                             

• Buffer A (extraction buffer): 15 ml
• Buffer B (10x denaturing buffer): 1.5 ml
• Buffer C (10x non-denaturing buffer): 1.5 ml
• 1.5 ml microfuge tube: 20
• Pestles for 1.5 ml tube: 2
• Filter Cartridge with Collection Tubes: 20
• Protein Extraction Powder: 2 g

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