Transmembrane Protein Extraction Reagent (tmPER-200)

Transmembrane Protein Extraction

The FIVEphoton Biochemicals Transmembrane Protein Extraction Reagent (tmPER-200TM) is a cell lysis-protein extraction buffer with proprietary ingredients that assist in the extraction and isolation of high molecular weight multiple-membrane spanning proteins that are otherwise poorly resolvable in standard cell lysis buffers due to aggregation tendency, lipid raft association or other insolubility issues. The Transmembrane Protein Extraction Reagent is designed to extract proteins with 4 or more transmembrane domains, yet will also effectively resolve less complex transmembrane proteins. Examples for the applicability of the FIVEphoton Biochemicals Transmembrane Protein Extraction Reagent are for enhanced resolution of ABC transporters, Ion Channels, Ion Exchangers and GPCRs in the downstream application of Western blotting. The Transmembrane Protein Extraction Reagent is applicable when RIPA buffer fails to extract and solubilize due to high molecular weight and aggregation tendency.


Transmembrane Protein Extraction Reagent: General Protocol

The researcher first employs techniques described in the protocol manual to limit endocytosis and lysosomal targeting that can otherwise result in proteolytic cleavage of cytoplasmic domains of multi-membrane spanning proteins. In the second step, the Transmembrane Protein Extraction Reagent, with added protease inhibitors, is added to dislodge cells from the cell culture dish and to dissolve the cell membrane. Following membrane dissolution, brief centrifugation is used to remove cellular debris from a supernatant fraction that contains extracted transmembrane proteins. The supernatant is added to Laemmli Sample Buffer, which is heated, but not boiled, prior to the resolution in SDS-PAGE gels. For Western blots, transfer takes place in a transfer buffer that has added SDS and reduced amounts of methanol. The transfer is allowed to proceed approximately 25% longer than typically used in Western blotting protocols.

1. Berberian G, Bollo M, Montich G, Roberts G, Degiorgis JA, et al. 2009. A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger. Biochim Biophys Acta 1788: 1255-62

2. Castro-Parodi M, Levi L, Dietrich V, Zotta E, Damiano AE. 2009. CFTR may modulate AQP9 functionality in preeclamptic placentas. Placenta 30: 642-8

3. Chen WY, Xu WM, Chen ZH, Ni Y, Yuan YY, et al. 2009. Cl- is required for HCO3- entry necessary for sperm capacitation in guinea pig: involvement of a Cl-/HCO3- exchanger (SLC26A3) and CFTR. Biol Reprod 80: 115-23

4. Gad A, Callender DL, Killeen E, Hudak J, Dlugosz MA, et al. 2009. Transient in utero disruption of cystic fibrosis transmembrane conductance regulator causes phenotypic changes in alveolar type II cells in adult rats. BMC Cell Biol 10: 24

5. Ghoshal A, Mukhopadhyay S, Demine R, Forgber M, Jarmalavicius S, et al. 2009. Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis. Glycoconj J 26: 675-89

6. Glaser SS, Gaudio E, Rao A, Pierce LM, Onori P, et al. 2009. Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium. Lab Invest 89: 456-69

7. Huang D, Gao Q, Guo L, Zhang C, Jiang W, et al. 2009. Isolation and identification of cancer stem-like cells in esophageal carcinoma cell lines. Stem Cells Dev 18: 465-73

8. Kang KW, Im YB, Go WJ, Han HK. 2009. C-myc amplification altered the gene expression of ABC- and SLC-transporters in human breast epithelial cells. Mol Pharm 6: 627-33