Transmembrane Protein Extraction Reagent (Part No. tmPER-50; Plasma Protein Isolation)

Transmembrane Protein Extraction Reagent

(Membrane Protein Isolation. Mammalian cells & plants cells)

Part: tmper-50: 30 ml volume.

Suitable for one hundred 10 cm cell culture dishes of confluent cells. Scalable to other quantities and sizes of culture plates.

Significantly higher solubilization and resolving ability for large transmembrane proteins compared to RIPA buffer.
Applicable to mammal and plant cell protein extraction. See product citations below.
Ideal for large multi-pass (4+ transmembrane domains) proteins that aggregate.
Contains a proprietary ingredient to extract TM proteins embedded in lipid rafts.
pH 7.4. 1X solution. 30ml volume.
Compatible with BCA assay.
Storage: 4^oC. Ships at ambient temperature.

Protocol

Transmembrane Protein Extraction Reagent (tmPER-50^TM ); Overview

The FIVEphoton Biochemicals Transmembrane Protein Extraction Reagent (tmPER-50^TM) is a cell lysis-protein extraction buffer with proprietary ingredients assisting in the isolation and solubility 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 include extraction and 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 resolve high molecular weight proteins with a tendency to aggregate.

Transmembrane Protein Extraction Reagent (tmPER-50^TM); General Protocol

The researcher first employs techniques to limit endocytosis and lysosomal targeting that may result in proteolytic cleavage of the cytoplasmic domains of multi-membrane spanning protein. In the second step, the Transmembrane Protein Extraction Reagent is added to dislodge cells from the cell culture dish and then to dissolve the cell membrane. Following membrane dissolution, brief centrifugation is used to remove cellular debris from a supernatant fraction that contains the 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 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.

An easy to follow protocol for cell lysis and preparation of samples for Western Blotting is included with the Transmembrane Protein Extraction Reagent, or can be downloaded in pdf format by clicking the manual tabs.

Icon Image: Application of the Transmembrane Protein Extraction Reagent for Western blot analysis of the complex multi-pass transmembrane protein CFTR. Western blot of deltaF508 and wild-type CFTR. First lane: Western blot of a 150 kD variant of a mutant ABC transporter showing the isoform with only core glycosylation. Second lane: Western blot of the wild-type variant of the same ABC transporter capturing both core and complex glycosylation. The mature-processed wild-type isoform with complex glycosylation shown on the right has a molecular mass of 170 kD and contains 12 transmembrane domains.

Volume Options for Transmembrane Protein Extraction Reagent

Available Volumes	Price	Part No.
30 ml	$180	TmPER-50
50 ml	$200	TmPER-100
100 ml	$260	TmPER-200

*Use Available Options menu to order volume options.

Product Citations

Animal Cells

K J Senthil Kumar, M Gokila Vani, Hen-Wen Hsieh, Chin-Chung Lin, Jiunn-Wang Liao, Pin-Ju Chueh, Sheng-Yang Wang (2019) MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling, Carcinogenesis, , bgz011, L ink to article
Downey, Anne Marie, Barbara F. Hales, and Bernard Robaire. "Zinc Transport Differs in Rat Spermatogenic Cells and Is Affected by Treatment with Cyclophosphamide." Biology of Reproduction (2016): biolreprod-116. Link to article
Kang, X., Lu, Z., Cui, C., Deng, M., Fan, Y., Dong, B., ... & Zhang, C. C. (2015). The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development. Nature cell biology. Link to article.
Dae-Gyun Ahn, Tanveer Sharif, Kenneth Chisholm, Devanand M Pinto, Shashi A Gujar & Patrick WK Lee (2015) Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response, Cell Cycle, 14:14, 2301-2310. Link to article.

Tissues

Li, M. H., Suchland, K. L., & Ingram, S. L. (2016). Compensatory activation of cannabinoid CB2 receptor inhibition of GABA release in the rostral ventromedial medulla (RVM) in inflammatory pain. Journal of Neuroscience, 1310-16. Link to article.

Plant Cells

Gerttula, S., Zinkgraf, M., Muday, G., Lewis, D., Ibatullin, F. M., Brumer, H., ... & Groover, A. (2015). Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus. The Plant Cell, tpc-15. Link to article
Wang, Y., Itaya, A., Zhong, X., Wu, Y., Zhang, J., van der Knaap, E., ... & Ding, B. (2011). Function and evolution of a microRNA that regulates a Ca2+-ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth. The Plant Cell, 23(9), 3185-3203. Link to article

Protocol

Representative References of High Molecular Weight Transmembrane Proteins

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

tmPER-50^TM is provided in 30 ml volume. (tmPER-100^TM; 50 ml. tmPER-200^TM; 100 ml)

Supplemental Products

Item

Protease Inhibitor Cocktail

Phosphatase Inhibitor Cocktail

Sodium Orthovanadate

Shipping: Ships at ambient temperature. International delivery available.

Storage: 4^oC

kw. PLASMA MEMBRANE PROTEIN, TRANSMEMBRANE, PLANT PROTEIN EXTRACTION REAGENT, MAMMALIAN CELL PROTEIN EXTRACTION, MEMBRANE LYSIS, WESTERN BLOT PROTEIN EXTRACTION

Category	Download Link
Protocol Manual	click here
MSDS	click here

CALCIUM PHOSPHATE TRANSFECTION KIT \| 200 10cm cell culture dishes \| Part No. CP-4 CP-4 (200 plates) $385.00	TRANSMEMBRANE PROTEIN EXTRACTION REAGENT (tmPER-100; Membrane Protein Isolation) TmPER-100 (150 plates) $200.00	Transmembrane Protein Extraction Reagent (tmPER-200; Plasma Protein Isolation) TmPER-200 (300 plates) $260.00	ECL Chemiluminescence Kit \| FIVEphoton Biochemicals \| PCD-250, PCD-500 \| High Sensitivity PCD-500 $179.95
SODIUM ORTHOVANADATE, ACTIVATED, 200mM SOLN \| Part ActVO-4 ActVO-4 Activated $145.00	RIPA Buffer (RIPA-50) RIPA-50 $82.00	PROTEASE INHIBITOR COCKTAIL BROAD SPECTRUM (100X; PI-1) PI-1 $115.95	PROTEASE INHIBITOR COCKTAIL w/ EDTA (100X, 1ml; PI-2) PI-2 $115.95
CHAPS IMMUNOPRECIPITATION (IP) AND LYSIS BUFFER For Tissues and Cells \| Part No. CIB-1 CIB-1 $175.00	Immunoprecipitation Buffer w/ Triton X-100 (TIB-1) TIB-1 $145.00	Tissue Protein Extraction Buffer (1X, 100 ml; Part No TEB-1) TEB-1 $125.00

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