Minute™ Plasma Membrane Protein Isolation Kit

Invent Biotechnologies Minute™ plasma membrane protein isolation kit is composed of optimized buffers and protein extraction filter cartridges with 2.0 ml collection tubes.
The kit is designed to rapidly isolate native total membrane proteins (organelle membrane proteins) and native plasma membrane proteins from cultured mammalian cells or tissues.

This kit can sequentially separate cellular components into four fractions: nuclei, cytosol, organelles and plasma membrane. Due to the use of protein extraction filter cartridges, the membrane protein isolation is simple, easy and user friendly with high yield. Unlike many commercial membrane preparation kits that require large amount of starting cells (5 millions and up). This kit offers wide range of starting cells (1-50 millions/sample). The buffers are detergent and EDTA free. A Dounce homogenizer or a tissue blender is not needed. The procedure can be completed in less than 45 min.

Applications

The kit is designed to rapidly isolate native membrane proteins from cultured cells or tissues for applications such as SDS-PAGE, immunoblottings, ELISA, IP, membrane protein structure analysis, 2-D gels, enzyme activity assays and other applications. This kit provides the most rapid method currently available for preparation of native membrane proteins.

Buffer Formulations: Proprietary

Kit components (50 preps)

1. 25 ml buffer A
2. 10 ml buffer B
3. 50 protein extraction filter cartridges
4. 50 collection tubes with cap5 4 plastic rods
6. Tissue dissociation beads

 A. SDS-PAGE profiles of total cell lysate (TC) and isolated plasma membrane proteins (PM) from human and rat cultured cells. Lane 1: Human lung cancer cell A549 total cell lysate; Lane 2: Isolated PM of A549; Lane 3: PM of rat REL-6TN cells; Lane 4: Total cell lysate of rat REL-6TN cells.
B. Western blottings: Proteins in A were transferred to nitrocellulose membrane and probed with anti-Na/K ATPase alpha1, a plasma membrane marker (Upstate, Clone 464.6)) and anti-lamin B1, a nuclear envelope marker (ab16048, abcam Cambridge, MA). The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD).
C. Densitometry measurement of Na/K ATPase alpha1 signals in B (TC vs. PM). Total cell lysates were extracted by Minute™ Denaturing Total Protein Extraction Kit (#SD-001, Invent Biotechnologies, Eden Prairie, MN).

Important Information:
1. Read the entire procedures carefully. Thaw buffer A and buffer B completely, invert the bottles a few times and place them on ice. Chill protein extraction filter cartridge with collection tube on ice prior to use.
2. All centrifugation steps should be performed at 4oC in a cold room or in a refrigerated mirocentrifuge.
3. To study protein phosphorylation, phosphatase inhibitors should be added to buffer A prior to use. The use of protease inhibitor cocktails is optional.
4. It is recommended to use BCA Protein Assay Kit for determination of protein concentration.

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