Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit
Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit is a novel and patented native PM protein isolation kit. The principle of isolation is: Cells/Tissues are first sensitized by buffer A then pass through a filter that allows cells to pass through with a zigzag path. The cell membranes are ruptured during the process and leave nuclei intact therefore the final PM prep is basically free of nuclear membrane and nuclear protein contamination. PM is separated from a mixture of un-ruptured cells (very small amount), nuclei, cytosol and organelles by subsequent differential centrifugation and density centrifugation without using ultracentrifugation. Since cell membranes are ruptured during the zigzag path when high speed centrifugal force is applied there is no need to use a homogenizer that are required by other kits. When a homogenizer is used in PM isolation, inconsistency is a major problem because variation in the duration of homogenization results in a different protein profile every time therefore resulting in a significant variation in final PM purity (inter-experiment variation). As a comparison, we use the same amount of starting cell, defined centrifugal force and pre-defined duration in every experiment and the result is much more consistent. The procedure can be completed in less than 45 min.
Price:$340 (50 preps)
Figure 1. 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).
Figure 2 A. SDS-PAGE profiles of isolated total membrane proteins from mouse tissues (T=Total Cell lysate, C=Cytosol Fraction, M= Total Membrane Fraction)
B. Proteins shown in A were transferred to a nitrocellulose membrane and probed with rabbit-anti mouse pan-cadherin (ab6529,abcam, Cambridge, MA), and anti-actin by Western blotting. The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD). Signals of pan-cadherin (a 130 kda plasma membrane marker) were significantly enhanced in total membrane protein fractions. Total cell lysates were extracted by MinuteTM Denaturing Total Protein Extraction Kit (SD-001, Invent Biotechnologies, Eden Prairie, MN).
Figure 3. A. SDS-PAGE profiles of total membrane protein fraction (TM) and isolated plasma membrane protein fraction from mouse tissues.
B. Western blotting of proteins in A were transferred to nitrocellulose membrane and probed with rabbit anti-cadherin (abcam, Cambridge, MA). The specific protein bands were visualized by a color metric substrate Opti-4CN (Bio-RAD).
C. Densitometry measurement of cadherin signals in B (TM vs. PM).
|Buffer A||25 ml||SM-005-A|
|Buffer B||10 ml||SM-005-B|
|Protein Extraction Filter Cartridges||50||P-001|
|Collection tubes with cap||50||P-002|
|Tissue dissociation beads||10 g||P-004|