Membrane Protein Extraction Kit

Application: Immunoprecipitation (IP)

Catalog no.: ABIN412488

Additional Information

Description: The Membrane Protein Extraction Kit provides optimized buffers and reagents for effective extraction of membrane proteins from mammalian tissues and cells. Unlike other available procedures that can only extract the total cellular membrane proteins (combinations of plasma and organelle membrane proteins), BioVision’s kit was designed to not only extract the total cellular membrane proteins, but also purify the plasma membrane proteins specifically. The procedure offers consistent yield and high purity (over 90%). Membrane proteins prepared using the kit can be utilized in a variety of applications, such as Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.

Application Details

Application Notes: Note: Some precipitation may occur after adding the Protease Inhibitor Cocktail. You may continue using the buffer or simply remove the precipitates by centrifugation). • The following protocol is described for extraction of ~5-10 x 10 8 cells. If more cells are used, scale up the volume proportionally. B. Extraction of Total Cellular Membrane Proteins: 1. Collect cells (5-10 x 10 8 ) by centrifugation at 600 x g for 5 minutes at 4 o C. For adherent cells, scrape cells in PBS and then spin down (3000 rpm for 5 minutes) to pellet cells. 2. Wash cells once with 1 ml of ice cold PBS. 3. Resuspend cells in 1 ml of the Homogenize Buffer Mix in an ice-cold Dounce homogenizer (Cat.# 1998-1). Homogenize cells on ice for 30-50 times. For tissue samples, homogenize tissues in 2-3 volume of the 1X Homogenize Buffer, until it is completed lysed (30-50 times). Efficient homogenization may depend on the cell type. To check the efficiency of the homogenization, pipette 2-3 μ l of the homogenized suspension onto a cover slip and observe under a microscope. A shiny ring around the nuclei indicates that cells are still intact. If 70-80 percent of the nuclei do not have the shiny ring, proceed to the next step. Otherwise, perform 10-30 additional passes. 4. Transfer the homogenate to a 1.5 ml microcentrifuge tube. Centrifuge in 700 g for 10 minutes at 4 o C. Collect supernatant and discard the pellet. 5. Transfer the supernatant to a new vial and centrifuge at 10,000g for 30 min at 4 o C. 6. Collect supernatant (This is Cytosol Fraction). The pellet is the total cellular membrane protein (containing proteins from both plasma membrane and cellular organelle membrane). You may stop here if you only need the total cellular membrane proteins. If you would like to further isolate the plasma membrane proteins specifically, continue with the following steps. C. Purification of Plasma Membrane Proteins: 7. Resuspend the total membrane proteins pellet in 200 μ l of the Upper Phase Solution. Add 200 μ l of the Lower Phase Solution. Mix well and incubate on ice for 5 minutes (Mark the tube as A). 8. Prepare a fresh phase tube without samples. Adding 200 μ l of Upper Phase Solution and 200 μ l of Lower Phase Solution (Mark the tube as B). 9. Centrifuge both A & B tubes in a microcentrifuge at 3500 rpm (1000 x g) for 5 minutes. 10. Carefully transfer the upper phase from tube A to a new tube (tube C), keep on ice. 11. To maximize the yield, extract the tube A lower phase again by adding 100 μ l of the Upper Phase Solution from tube B. Mix well and centrifuge at 3500 rpm (1000 g) for 5 minutes. 12. Carefully collect the upper phase. Combine with the upper phase from Step 10 (tube C). Extract the combined upper phase by adding 100 μ l of the Lower Phase Solution from tube B, Mix well and centrifuge at 3500 rpm (1000 g) for 5 minutes. 13. Carefully collect the upper phase. Dilute the upper phase in 5 volume of water. Keep on ice for 5 minutes. 14. Spin at top speed at a microcentrifuge tube for 10 minutes at 4 o C. Remove the supernatant. The pellet is the plasma membrane protein. 15. Store the plasma membrane proteins at –70 o C for further studies. The membrane fraction can be dissolved in 0.5% Triton X-100 in PBS or other buffers before use. Generally 1-100 μ g plasma membrane proteins can be obtained.

Restrictions: For Research Use only

Publications

Publications: Wu, Zhou, Luo: "Geranylgeranyltransferase I is essential for dendritic development of cerebellar Purkinje cells." in: Molecular brain, Vol. 3, pp. 18, 2010 (PubMed).

Banerjee, Wang, Alzamora et al.: "SGLT1, a novel cardiac glucose transporter, mediates increased glucose uptake in PRKAG2 cardiomyopathy." in: Journal of molecular and cellular cardiology, 2010 (PubMed).

Ghebeh, Lehe, Barhoush et al.: "Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule." in: Breast cancer research : BCR, Vol. 12, Issue 4, pp. R48, 2010 (PubMed).

Moon, Lee, Kang et al.: "Dietary monounsaturated fatty acids but not saturated fatty acids preserve the insulin signaling pathway via IRS-1/PI3K in rat skeletal muscle." in: Lipids, Vol. 45, Issue 12, pp. 1109-16, 2010 (PubMed).

Zhang, Liu, Liu et al.: "Epithelial-mesenchymal transition of rat peritoneal mesothelial cells via Rhoa/Rock pathway." in: In vitro cellular & developmental biology. Animal, 2010 (PubMed).

Turdi, Kandadi, Zhao et al.: "Deficiency in AMP-Activated Protein Kinase Exaggerates High Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction." in: Journal of molecular and cellular cardiology, 2010 (PubMed).

Wegiel, Gallo, Csizmadia et al.: "Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase." in: Proceedings of the National Academy of Sciences of the United States of America, 2011 (PubMed).

Prat, Blanchon, Borel et al.: "Ontogeny of Aquaporins in Human Fetal Membranes." in: Biology of reproduction, 2011 (PubMed).