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Membrane Protein Extraction and Isolation |
Membrane protein extraction and isolation is critical to understanding the structural and functional role of proteins within biological systems. Membrane proteins play crucial roles in various biological processes, serving as key players in cell signaling, transport, and communication between cells and their environment. However, these proteins embedded with the cell membranes lipid bilayer, presents unique challenges in their separation from other cellular components due to their hydrophobic nature and localization. Membrane protein extraction and isolation methods utilize strategies to obtain pure and functional membrane proteins for further analysis and research applications.
Thermo Fisher Scientific offers specialized membrane protein extraction and isolation reagents, kits, polymers, and detergents that are optimized for high protein yield, preserving protein function, minimizing contamination, and helping ensure compatibility with downstream applications. Choose from our comprehensive range of membrane protein extraction and isolation products based on your specific isolation needs.
On this page:
Strategies for extraction and stabilization
Pre-formulated reagents and kits | Detergents | Polymers |
|---|---|---|
| ✓ Traditional protein extraction method ✓ Optimized buffers and reagents ✓ No upfront screening • May need to buffer exchange downstream | ✓ Maintains protein-protein interactions, protein activity and function, and protein structure | ✓ Detergent-free method for extracting proteins and surrounding lipids • Need to ensure native conformation • Requires screening/research to confirm fit for purpose |
Learn more: Membrane protein expression and purification
Choose the appropriate membrane protein extraction method
 |  |  | |
| GPCR Extraction and Stabilization Reagent | Mem-PER Plus Membrane Protein Extraction Kit | Cell Surface Protein Biotinylation and Isolation Kit | |
| Membrane protein enrichment | G Protein-Coupled Receptors (GPCR) | Plasma membrane proteins | Integral and membrane associated proteins |
| Protein functionality | Maintains structural integrity as measured by immunoprecipitation and receptor ligand binding assays | Enrichment compatible with western blotting | Enrichment compatible with western blotting and MS applications |
| Compatible sample types | Tissues and cultured mammalian cells | Tissues and cultured mammalian cells | Cultured mammalian cells |
| Cytosolic contamination | Whole cell lysate | Less than 5% contamination of cytosolic proteins | Less than 20% contamination of cytosolic proteins |
| Sample processing time | 1–2 hours | 1 hour | 2 hours |
| Mechanical disruption required? | Yes, for tissue | Yes, for tissue | No |
| Amount of sample processed | 100 samples with 107 cells or 100 samples with 50–100 mg tissue | 50 samples with 5 million cells or 25 samples of 20–40 mg tissue per kit | 8 experiments, with two confluent 15 cm dishes or four T75 flasks |
| Compatible protein assays | BCA Protein Assays | BCA Protein Assays, Detergent-Compatible Bradford | Lysis buffer is compatible with BCA and Rapid Gold BCA; elution buffer is not compatible |
| Downstream compatibility | Western blot, IP, protein purification, radio-ligand binding assays | IP, western blot, ELISA, amine reactive labeling | Western blot, ELISA, mass spectrometry analysis |
| Protease/phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, both |
| Available size(s) | 100 mL | 300 mL | 8 samples |
| User guide | User Guide: GPCR Extraction and Stabilization Reagent | User Guide: Mem-PER Plus Membrane Protein Extraction Kit | User Guide: Pierce Cell Surface Protein Biotinylation and Isolation Kit |
Reagent-based membrane protein extraction and isolation
Reagent-based lysis has replaced traditional physical lysis as the method of choice for membrane disruption and extraction of membrane proteins. Reagent-based lysis methods do not require expensive, cumbersome equipment and protocols that are difficult to implement. The Thermo Scientific membrane protein extraction and membrane protein isolation reagents consist of optimized concentrations of detergents, buffers, salts, and reducing agents developed for particular species and types of cells. Reagents also have the added benefits of both lysing and solubilizing effects. Our membrane protein isolation and membrane protein extraction kits are optimized specifically for your needs to isolate high-quality protein samples essential to completing successful downstream applications.
Features:
- Extraction and isolation—produces minimal cross-contamination (typically less than 10%) of cytosolic protein into the membrane protein fraction
- Cells or tissues—effective for extraction from cultured mammalian cells and mammalian tissues
- Downstream compatibility—analyze membrane protein extracts by SDS-PAGE, western blotting, immunoprecipitation, and protein assays
- Fast and simple—isolation of membrane proteins in approximately one hour
- No special equipment required—only a benchtop microcentrifuge, tubes, homogenizers, and pipettors are needed
Membrane protein isolation kits
Extraction and stabilization of GPCR’s
The GPCR Extraction and Stabilization Reagent is an easy-to-use solution that extracts and stabilizes G-couples protein receptors (GPCR’s) and other membrane-associated protein from cells and tissues in 1–2 hours (Figure 1). This all-in-one reagent stabilizes the receptor by encapsulating it in a detergent micelle that maintains the structural and functional integrity of the protein (Figure 2). This stabilization process also allows for more flexibility for receptor analysis by enabling extended storage of the extracted protein at 4°C for up to one week and at –20°C for up to one month.
Application note:Extraction and Purification of Functional G Protein-Coupled Receptors from Expression System
Poster:Effective solubilization and stabilization of functional G protein-coupled receptors
Click image to enlarge
Figure 1. Preparation of lysates with soluble and stable GPCRs.
Click image to enlarge
Figure 2. Preserve functionality of receptors in cells and tissues. Extracts from GeneBLAzer expression cell lines or mouse brain tissue were prepared using the GPCR Extraction and Stabilization Reagent to assess activity using radioligand binding assays. Extracts (400–600 µg) containing Muscarinic Acetylcholine Receptor 3 (M3) and serotonin receptor (5HT1A) (GeneBLAzer expression cell lines) or adenosine A2a receptor (ADORA2A) (mouse brain tissue) were then incubated with H3-4-DAMP, H3-propanolol, or H3-adenosine (PerkinElmer) alone or in a competition reaction of cold H3-ligand for 75 minutes at room temperature. Free radioligand was then removed and samples were then analyzed using a TRI-CARB 2000 TR scintillation counter.
Extraction and enrichment of integral membrane proteins
MEM-PER Plus Membrane Protein Extraction Kit allows for solubilization and enrichment of integral membrane proteins and membrane-associated proteins. This membrane protein isolation kit offers a user-friendly alternative to traditional protein isolation techniques, utilizing a straightforward benchtop microcentrifuge procedure (Figure 3).
Application note:Efficient Mammalian Membrane Protein Extraction
Click image to enlarge
Figure 3. Protocol summary for the Mem-PER Plus Membrane Protein Extraction Kit.
Membrane protein extraction and enrichment can be obtained from adherent and suspension cells as well as hard and soft tissues (Figure 4). The extraction efficiencies and yields of integral membrane proteins will depend on the cell type and the number of transmembrane domains. Typically, proteins with one or two transmembrane domains have a high extraction efficiency (up to 90%), while cross-contamination of cytosolic proteins into the membrane fraction is typically minimal (less than 10%).
Figure 4. Isolation and enrichment of membrane proteins from different tissues. Membrane proteins were isolated from frozen mouse heart or brain (30 mg) following the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane and cytosolic fractions (10 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blots were done using the Thermo Scientific Fast Western Rabbit Dura Kit and primary antibodies diluted 1:1000. Images were generated using the Thermo Scientific myECL Imager.
Biotinylation and enrichment of plasma membrane proteins
The Cell Surface Biotinylation and Isolation Kit biotinylates, solubilizes, and enriches plasma membrane proteins. This membrane protein isolation kit offers a less harsh alternative to traditional protein isolation techniques that can result in denatured and contaminated plasma membrane proteins. The cell surface biotinylation and isolation kit is easy to use with a reduced number of steps which reduces processing time, and reagent formulations to improve enrichment and extraction while minimizing intracellular protein contamination. This procedure involves the biotinylation of proteins using a thiol-cleavable amine-reactive biotinylation reagent, EZ-Link Sulfo-NHS-SS-Biotin. Cells are then lysed, and labeled proteins are captured with NeutrAvidin Agarose and eluted with dithiothreitol (DTT) to reduce disulfide bonds in the biotin label (Figure 5).
Poster: Proteomic analysis of cell surface proteins with improved specificity of enrichment
Click image to enlarge
Figure 5. Protocol summary for the Cell Surface Protein Biotinylation and Isolation Kit.
The cell surface biotinylation and isolation kit is compatible with a range of adherent and suspension cell lines. It also allows for the comparative protein expression analysis in treated versus non-treated cells as well as different cell lines. Selectivity and efficiency of isolated membrane protein can be confirmed by western blot (Figure 6) and LC-MS analysis (Figure 7).
Click image to enlarge
Figure 6. Western blot analysis of enriched cell surface proteins. Samples were prepared in duplicate from two cell lines using the Cell Surface Protein Biotinylation and Isolation Kit. Flow-through (F), elution (E), and bead boil (B) were normalized by volume and analyzed by western blot for cell surface proteins (EGFR, CD55, cadherin, and integrin α5) or intracellular proteins (HSP90, actin, calnexin, and β-tubulin). The blots were imaged using the iBright FL Imaging System. The data demonstrates effective enrichment of cell surface proteins with minimal contamination of intracellular proteins.
Click image to enlarge
Figure 7. Classification of cell surface protein type in the top 50 most abundant proteins. Cell surface proteins were enriched from three mammalian cell lines, in duplicate, and then analyzed by LC-MS. Raw data was searched using Proteome Discoverer Software 2.2. The top 50 most abundant proteins were identified using the UniProt Knowledgebase. Cell surface proteins were classified as single- or multi-spanning (6–17 spans) transmembrane (TM) proteins, glycosylphosphatidylinositol (GPI) anchored proteins, or extracellular matrix (ECM) proteins. A similar distribution was observed in the three cell lines.
Non-denaturing detergents are excellent for membrane protein isolation as they maintain protein-protein interactions, protein activity and function, and protein structure. These types of detergents include non-ionic and zwitterionic. Non-ionic detergents are milder and are recommended to be used when maintaining protein interactions and activity is critical. Zwitterionic detergents are stronger and usually result in better membrane solubilization. See the different characteristics of non-denaturing detergents in the table below.
Characteristics of membrane protein isolation detergents
| Nonionic | Zwitterionic | |
|---|---|---|
| Maltosides | Glucosides | |
|
|
|
| When choosing a detergent for membrane protein extraction, it is important to remember the detergent best for solubilization may not be the best for your application. | ||
Learn more about non-ionic versus ionic, and non-denaturing versus denaturing detergents and see other detergents for protein solubilization
Matoside detergents | |||||||
|---|---|---|---|---|---|---|---|
Maltosides are highly efficient at extracting and stabilizing hydrophobic membrane proteins. They assist in the purification and characterization of membrane proteins as such can be used in structural and functional analysis. | |||||||
| n-Dodecyl-β-Maltoside Detergent (DDM) | Lauryl Maltose Neopentyl Glycol (LMNG) | n-Undecyl-β-D-maltopyranoside (UDM) | n-Octyl-β-D-Maltopyranoside (OM) | n-Nonyl-β-Maltoside (NM) | |||
| Description type | Non-ionic | ||||||
| Functional properties | Used most often for hydrophobic membrane proteins. | Ideal for integral membrane proteins. | Forms stronger interactions with hydrophobic regions of membranes proteins. | Flexible for use in a wide range of experimental conditions, buffer systems, and pH rangers. | Forms stable micelles at lower concentrations important for protein stability and integrity. | ||
| Dialyzable | No | Yes | Yes | Yes | Yes | ||
| Aggregation number* | 98 average (70–140 range) | Unknown | ~71 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ~47 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ~55 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ||
| MW | 510.6 | 1005.2 | 496.6 | 454.4 | 468.5 | ||
| CMC %w/v (mM) | 0.009% (0.17) in water | 0.001% (~0.01) | 0.03% (~0.59) | 0.89% (~19.5) in 100 mM NaCl, 20 mM HEPES pH 7.5 | 0.28% (~6) | ||
| Compatible with Detergent Removal Resin | Yes | No | Yes | No | Yes | ||
| Available size(s) | 1 g 5 g | 1 g (solid) 10 x 1 mL (liquid) | 1 g 5 g | 1 g 5 g | 1 g 5 g | ||
See all maltoside detergents | |||||||
| Cyclohexyl maltoside detergents | |||||||
| Cyclohexyl maltosides are designed for the extraction of intact complexes. They can be used with protein purification, crystallization, spectroscopy, and structural studies. | |||||||
| 5-Cyclohexyl-1-Pentyl-β-D-Maltoside | 6-Cyclohexyl-1-Hexyl-β-D-Maltoside | 7-Cyclohexyl-1-Heptyl-β-D-Maltoside | |||||
| Description type | Non-ionic | ||||||
| Functional properties | Ideal for membrane protein complexes, solubilization efficiency depends on specific protein, structural features, and experimental conditions. | Increased hydrophobicity may result in enhanced solubilization of higher hydrophobic regions or multiple transmembrane domains. Well suited for structural biology studies. | Provides improved stability, maintaining structural integrity and functionality. Well suited for structural biology studies. | ||||
| Dialyzable | Yes | Yes | Yes | ||||
| Aggregation number* | ~47 | ~91 | ~150 | ||||
| MW | 494.5 | 508.5 | 522.5 | ||||
| CMC %w/v (mM) | 0.12% (~2.4–5.0) | 0.028% (~0.56) | 0.0099% (~0.19) | ||||
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | ||||
| Available size(s) | 1 g 5 g | 1 g 5 g | 1 g 5 g | ||||
See all cyclohexyl maltoside detergents | |||||||
*Aggregation number is the number of molecules per micelle
| Glucoside and other non-ionic detergents | ||||
|---|---|---|---|---|
| Glucosides are gentler detergents that enhance solubilization of hydrophobic biomolecules. They can be used with protein purification, crystallization, and structural studies. Tetraethylene Glycol Monooctyl Ether (C8E4) is good for solubilization of sensitive biomolecules. It can be used in native mass spectrometry. | ||||
| n-Nonyl-β-D-glucoside (NG) | Octyl-β-Glucoside Detergent (OG) | Octylthioglucoside (OTG) | Tetraethylene Glycol Monooctyl Ether (C8E4) | |
| Description type | Non-ionic | |||
| Functional properties | Contains a longer alkyl chain resulting in higher hydrophobicity, enhancing solubilization and improving extraction of membrane proteins. | Widely used for membrane protein solubilization Advantageous for application such as protein purification and structural studies since they provide better solubility and reduced downstream interference. | Effective for cell lysis and nondenaturing protein solubilization Useful for applications where enzyme stability is desired since it can remain stable in the presence of beta-D-glucoside glucohydrolase enzymes. | Good for solubility in both aqueous and organic solvents Ideal for sensitive biomolecules, and membrane proteins or protein complexes used for MS. |
| Dialyzable | Yes | Yes | Yes | Yes |
| Aggregation number* | Unknown | 27 | Unknown | ~82 |
| MW | 308.4 | 292.4 | 44 | 306.5 |
| CMC %w/v (mM) | 0.20% (6.5) | 0.67–0.73% (23-25) | 0.28% (9) | 0.25% (~8) |
| Cloud point (°C) | Unknown | >100 | >100 | Unknown |
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | Yes |
| Available size(s) | 1 g 5 g | 5 g | 5 g | 10 mL |
| See all glucoside detergents | ||||
* Aggregation number is the number of molecules per micelle
| Zwitterionic detergents | ||||
|---|---|---|---|---|
| Tetraethylene Glycol Monooctyl Ether (C8E4), Lauryldimethylamine-N-oxide (LDAO), and Fos-Choline detergents can all be used for protein purification and structural studies. LDAO are ideal for MS and crystallization studies; while fos-choline is also used for membrane protein research. | ||||
| Lauryldimethylamine-N-oxide (LDAO) | Fluorinated Fos-Choline-8 | Fos-choline-12 | Fos-choline-15 | |
| Description type | Zwitterionic | |||
| Functional properties | Ideal for the study of intact membrane protein structure and non-covalent interactions. | Efficient at solubilization of challenging and delicate membrane proteins. | ||
| Ideal for sensitive membrane proteins, minimizes potential cytotoxic effects such as denaturation and aggregation. | Optimal for wide range of membrane protein types by maintaining native conformation and functionality of proteins. | Increased hydrophobicity results in enhanced solubilization and stabilization of larger hydrophobic regions or transmembrane domains. | ||
| Dialyzable | Yes | Yes | Yes | Yes |
| Aggregation number* | ~76 | Unknown | ~54 | ~131 |
| MW | 229.4 | 529.2 | 351.5 | 393.5 |
| CMC % w/v (mM) | 0.023% (~1-2) | -- (~2.9) | 0.047% (1.5) | 0.0027% (0.07) |
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | No |
| Available size(s) | 1 g 5 g | 1 g 5 g | 1 g 5 g | 1 g 5 g |
| See all fos-choline detergents | ||||
* Aggregation number is the number of molecules per micelle
Not worried about maintaining protein function? See our detergents for protein solubilization
Polymers for detergent-free membrane protein isolation
Polymers represents a cutting-edge approach to membrane protein extraction, offering a detergent-free method for isolating membrane proteins along with their surrounding lipid bilayer. This innovative technology provides researchers with a powerful tool for studying membrane proteins in their native lipid environment, potentially leading to more accurate structural and functional analyses.
Like detergents, it is important to understand polymer characteristics in order to determine the best polymer for membrane protein target and intended experimental goal. A recent publication highlights the importance of selecting a polymer that effectively interacts with the specific lipid environment to maintain protein stability and functionality (1).
Characteristics of membrane protein extraction polymers
| Attribute | Unit | SMALP® 200 | SMALP® 300 |
|---|---|---|---|
| Polymer styrene-to-maleic anhydride ratio | n:m | 2:1 | 3:1 |
| Molecular weight | Da | 6,500 | 10,000 |
| Dry solids content | % w/w | 20 | 20 |
| Solvent | | Water | Water |
| pH | | 7.5 | 8.0 |
| Absolute viscosity | mPaꞏs | ≤ 5,000 | ≤ 5,000 |
| See all SMALP 200 polymers | See all SMALP 300 polymers | ||
| | | |
| GPCR Extraction and Stabilization Reagent | Mem-PER Plus Membrane Protein Extraction Kit | Cell Surface Protein Biotinylation and Isolation Kit | |
| Membrane protein enrichment | G Protein-Coupled Receptors (GPCR) | Plasma membrane proteins | Integral and membrane associated proteins |
| Protein functionality | Maintains structural integrity as measured by immunoprecipitation and receptor ligand binding assays | Enrichment compatible with western blotting | Enrichment compatible with western blotting and MS applications |
| Compatible sample types | Tissues and cultured mammalian cells | Tissues and cultured mammalian cells | Cultured mammalian cells |
| Cytosolic contamination | Whole cell lysate | Less than 5% contamination of cytosolic proteins | Less than 20% contamination of cytosolic proteins |
| Sample processing time | 1–2 hours | 1 hour | 2 hours |
| Mechanical disruption required? | Yes, for tissue | Yes, for tissue | No |
| Amount of sample processed | 100 samples with 107 cells or 100 samples with 50–100 mg tissue | 50 samples with 5 million cells or 25 samples of 20–40 mg tissue per kit | 8 experiments, with two confluent 15 cm dishes or four T75 flasks |
| Compatible protein assays | BCA Protein Assays | BCA Protein Assays, Detergent-Compatible Bradford | Lysis buffer is compatible with BCA and Rapid Gold BCA; elution buffer is not compatible |
| Downstream compatibility | Western blot, IP, protein purification, radio-ligand binding assays | IP, western blot, ELISA, amine reactive labeling | Western blot, ELISA, mass spectrometry analysis |
| Protease/phosphatase inhibitors recommended? | Yes, both | Yes, both | Yes, both |
| Available size(s) | 100 mL | 300 mL | 8 samples |
| User guide | User Guide: GPCR Extraction and Stabilization Reagent | User Guide: Mem-PER Plus Membrane Protein Extraction Kit | User Guide: Pierce Cell Surface Protein Biotinylation and Isolation Kit |
Reagent-based membrane protein extraction and isolation
Reagent-based lysis has replaced traditional physical lysis as the method of choice for membrane disruption and extraction of membrane proteins. Reagent-based lysis methods do not require expensive, cumbersome equipment and protocols that are difficult to implement. The Thermo Scientific membrane protein extraction and membrane protein isolation reagents consist of optimized concentrations of detergents, buffers, salts, and reducing agents developed for particular species and types of cells. Reagents also have the added benefits of both lysing and solubilizing effects. Our membrane protein isolation and membrane protein extraction kits are optimized specifically for your needs to isolate high-quality protein samples essential to completing successful downstream applications.
Features:
- Extraction and isolation—produces minimal cross-contamination (typically less than 10%) of cytosolic protein into the membrane protein fraction
- Cells or tissues—effective for extraction from cultured mammalian cells and mammalian tissues
- Downstream compatibility—analyze membrane protein extracts by SDS-PAGE, western blotting, immunoprecipitation, and protein assays
- Fast and simple—isolation of membrane proteins in approximately one hour
- No special equipment required—only a benchtop microcentrifuge, tubes, homogenizers, and pipettors are needed
Membrane protein isolation kits
Extraction and stabilization of GPCR’s
The GPCR Extraction and Stabilization Reagent is an easy-to-use solution that extracts and stabilizes G-couples protein receptors (GPCR’s) and other membrane-associated protein from cells and tissues in 1–2 hours (Figure 1). This all-in-one reagent stabilizes the receptor by encapsulating it in a detergent micelle that maintains the structural and functional integrity of the protein (Figure 2). This stabilization process also allows for more flexibility for receptor analysis by enabling extended storage of the extracted protein at 4°C for up to one week and at –20°C for up to one month.
Application note:Extraction and Purification of Functional G Protein-Coupled Receptors from Expression System
Poster:Effective solubilization and stabilization of functional G protein-coupled receptors
Click image to enlarge
Figure 1. Preparation of lysates with soluble and stable GPCRs.
Click image to enlarge
Figure 2. Preserve functionality of receptors in cells and tissues. Extracts from GeneBLAzer expression cell lines or mouse brain tissue were prepared using the GPCR Extraction and Stabilization Reagent to assess activity using radioligand binding assays. Extracts (400–600 µg) containing Muscarinic Acetylcholine Receptor 3 (M3) and serotonin receptor (5HT1A) (GeneBLAzer expression cell lines) or adenosine A2a receptor (ADORA2A) (mouse brain tissue) were then incubated with H3-4-DAMP, H3-propanolol, or H3-adenosine (PerkinElmer) alone or in a competition reaction of cold H3-ligand for 75 minutes at room temperature. Free radioligand was then removed and samples were then analyzed using a TRI-CARB 2000 TR scintillation counter.
Extraction and enrichment of integral membrane proteins
MEM-PER Plus Membrane Protein Extraction Kit allows for solubilization and enrichment of integral membrane proteins and membrane-associated proteins. This membrane protein isolation kit offers a user-friendly alternative to traditional protein isolation techniques, utilizing a straightforward benchtop microcentrifuge procedure (Figure 3).
Application note:Efficient Mammalian Membrane Protein Extraction
Click image to enlarge
Figure 3. Protocol summary for the Mem-PER Plus Membrane Protein Extraction Kit.
Membrane protein extraction and enrichment can be obtained from adherent and suspension cells as well as hard and soft tissues (Figure 4). The extraction efficiencies and yields of integral membrane proteins will depend on the cell type and the number of transmembrane domains. Typically, proteins with one or two transmembrane domains have a high extraction efficiency (up to 90%), while cross-contamination of cytosolic proteins into the membrane fraction is typically minimal (less than 10%).
Figure 4. Isolation and enrichment of membrane proteins from different tissues. Membrane proteins were isolated from frozen mouse heart or brain (30 mg) following the Mem-PER Plus Membrane Protein Extraction Kit protocol. Membrane and cytosolic fractions (10 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blots were done using the Thermo Scientific Fast Western Rabbit Dura Kit and primary antibodies diluted 1:1000. Images were generated using the Thermo Scientific myECL Imager.
Biotinylation and enrichment of plasma membrane proteins
The Cell Surface Biotinylation and Isolation Kit biotinylates, solubilizes, and enriches plasma membrane proteins. This membrane protein isolation kit offers a less harsh alternative to traditional protein isolation techniques that can result in denatured and contaminated plasma membrane proteins. The cell surface biotinylation and isolation kit is easy to use with a reduced number of steps which reduces processing time, and reagent formulations to improve enrichment and extraction while minimizing intracellular protein contamination. This procedure involves the biotinylation of proteins using a thiol-cleavable amine-reactive biotinylation reagent, EZ-Link Sulfo-NHS-SS-Biotin. Cells are then lysed, and labeled proteins are captured with NeutrAvidin Agarose and eluted with dithiothreitol (DTT) to reduce disulfide bonds in the biotin label (Figure 5).
Poster: Proteomic analysis of cell surface proteins with improved specificity of enrichment
Click image to enlarge
Figure 5. Protocol summary for the Cell Surface Protein Biotinylation and Isolation Kit.
The cell surface biotinylation and isolation kit is compatible with a range of adherent and suspension cell lines. It also allows for the comparative protein expression analysis in treated versus non-treated cells as well as different cell lines. Selectivity and efficiency of isolated membrane protein can be confirmed by western blot (Figure 6) and LC-MS analysis (Figure 7).
Click image to enlarge
Figure 6. Western blot analysis of enriched cell surface proteins. Samples were prepared in duplicate from two cell lines using the Cell Surface Protein Biotinylation and Isolation Kit. Flow-through (F), elution (E), and bead boil (B) were normalized by volume and analyzed by western blot for cell surface proteins (EGFR, CD55, cadherin, and integrin α5) or intracellular proteins (HSP90, actin, calnexin, and β-tubulin). The blots were imaged using the iBright FL Imaging System. The data demonstrates effective enrichment of cell surface proteins with minimal contamination of intracellular proteins.
Click image to enlarge
Figure 7. Classification of cell surface protein type in the top 50 most abundant proteins. Cell surface proteins were enriched from three mammalian cell lines, in duplicate, and then analyzed by LC-MS. Raw data was searched using Proteome Discoverer Software 2.2. The top 50 most abundant proteins were identified using the UniProt Knowledgebase. Cell surface proteins were classified as single- or multi-spanning (6–17 spans) transmembrane (TM) proteins, glycosylphosphatidylinositol (GPI) anchored proteins, or extracellular matrix (ECM) proteins. A similar distribution was observed in the three cell lines.
Non-denaturing detergents are excellent for membrane protein isolation as they maintain protein-protein interactions, protein activity and function, and protein structure. These types of detergents include non-ionic and zwitterionic. Non-ionic detergents are milder and are recommended to be used when maintaining protein interactions and activity is critical. Zwitterionic detergents are stronger and usually result in better membrane solubilization. See the different characteristics of non-denaturing detergents in the table below.
Characteristics of membrane protein isolation detergents
| Nonionic | Zwitterionic | |
|---|---|---|
| Maltosides | Glucosides | |
|
|
|
| When choosing a detergent for membrane protein extraction, it is important to remember the detergent best for solubilization may not be the best for your application. | ||
Learn more about non-ionic versus ionic, and non-denaturing versus denaturing detergents and see other detergents for protein solubilization
| Matoside detergents | |||||||
|---|---|---|---|---|---|---|---|
Maltosides are highly efficient at extracting and stabilizing hydrophobic membrane proteins. They assist in the purification and characterization of membrane proteins as such can be used in structural and functional analysis. | |||||||
| n-Dodecyl-β-Maltoside Detergent (DDM) | Lauryl Maltose Neopentyl Glycol (LMNG) | n-Undecyl-β-D-maltopyranoside (UDM) | n-Octyl-β-D-Maltopyranoside (OM) | n-Nonyl-β-Maltoside (NM) | |||
| Description type | Non-ionic | ||||||
| Functional properties | Used most often for hydrophobic membrane proteins. | Ideal for integral membrane proteins. | Forms stronger interactions with hydrophobic regions of membranes proteins. | Flexible for use in a wide range of experimental conditions, buffer systems, and pH rangers. | Forms stable micelles at lower concentrations important for protein stability and integrity. | ||
| Dialyzable | No | Yes | Yes | Yes | Yes | ||
| Aggregation number* | 98 average (70–140 range) | Unknown | ~71 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ~47 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ~55 in 100 mM NaCl, 20 mM HEPES pH 7.5 | ||
| MW | 510.6 | 1005.2 | 496.6 | 454.4 | 468.5 | ||
| CMC %w/v (mM) | 0.009% (0.17) in water | 0.001% (~0.01) | 0.03% (~0.59) | 0.89% (~19.5) in 100 mM NaCl, 20 mM HEPES pH 7.5 | 0.28% (~6) | ||
| Compatible with Detergent Removal Resin | Yes | No | Yes | No | Yes | ||
| Available size(s) | 1 g 5 g | 1 g (solid) 10 x 1 mL (liquid) | 1 g 5 g | 1 g 5 g | 1 g 5 g | ||
See all maltoside detergents | |||||||
| Cyclohexyl maltoside detergents | |||||||
| Cyclohexyl maltosides are designed for the extraction of intact complexes. They can be used with protein purification, crystallization, spectroscopy, and structural studies. | |||||||
| 5-Cyclohexyl-1-Pentyl-β-D-Maltoside | 6-Cyclohexyl-1-Hexyl-β-D-Maltoside | 7-Cyclohexyl-1-Heptyl-β-D-Maltoside | |||||
| Description type | Non-ionic | ||||||
| Functional properties | Ideal for membrane protein complexes, solubilization efficiency depends on specific protein, structural features, and experimental conditions. | Increased hydrophobicity may result in enhanced solubilization of higher hydrophobic regions or multiple transmembrane domains. Well suited for structural biology studies. | Provides improved stability, maintaining structural integrity and functionality. Well suited for structural biology studies. | ||||
| Dialyzable | Yes | Yes | Yes | ||||
| Aggregation number* | ~47 | ~91 | ~150 | ||||
| MW | 494.5 | 508.5 | 522.5 | ||||
| CMC %w/v (mM) | 0.12% (~2.4–5.0) | 0.028% (~0.56) | 0.0099% (~0.19) | ||||
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | ||||
| Available size(s) | 1 g 5 g | 1 g 5 g | 1 g 5 g | ||||
See all cyclohexyl maltoside detergents | |||||||
*Aggregation number is the number of molecules per micelle
| Glucoside and other non-ionic detergents | ||||
|---|---|---|---|---|
| Glucosides are gentler detergents that enhance solubilization of hydrophobic biomolecules. They can be used with protein purification, crystallization, and structural studies. Tetraethylene Glycol Monooctyl Ether (C8E4) is good for solubilization of sensitive biomolecules. It can be used in native mass spectrometry. | ||||
| n-Nonyl-β-D-glucoside (NG) | Octyl-β-Glucoside Detergent (OG) | Octylthioglucoside (OTG) | Tetraethylene Glycol Monooctyl Ether (C8E4) | |
| Description type | Non-ionic | |||
| Functional properties | Contains a longer alkyl chain resulting in higher hydrophobicity, enhancing solubilization and improving extraction of membrane proteins. | Widely used for membrane protein solubilization Advantageous for application such as protein purification and structural studies since they provide better solubility and reduced downstream interference. | Effective for cell lysis and nondenaturing protein solubilization Useful for applications where enzyme stability is desired since it can remain stable in the presence of beta-D-glucoside glucohydrolase enzymes. | Good for solubility in both aqueous and organic solvents Ideal for sensitive biomolecules, and membrane proteins or protein complexes used for MS. |
| Dialyzable | Yes | Yes | Yes | Yes |
| Aggregation number* | Unknown | 27 | Unknown | ~82 |
| MW | 308.4 | 292.4 | 44 | 306.5 |
| CMC %w/v (mM) | 0.20% (6.5) | 0.67–0.73% (23-25) | 0.28% (9) | 0.25% (~8) |
| Cloud point (°C) | Unknown | >100 | >100 | Unknown |
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | Yes |
| Available size(s) | 1 g 5 g | 5 g | 5 g | 10 mL |
| See all glucoside detergents | ||||
* Aggregation number is the number of molecules per micelle
| Zwitterionic detergents | ||||
|---|---|---|---|---|
| Tetraethylene Glycol Monooctyl Ether (C8E4), Lauryldimethylamine-N-oxide (LDAO), and Fos-Choline detergents can all be used for protein purification and structural studies. LDAO are ideal for MS and crystallization studies; while fos-choline is also used for membrane protein research. | ||||
| Lauryldimethylamine-N-oxide (LDAO) | Fluorinated Fos-Choline-8 | Fos-choline-12 | Fos-choline-15 | |
| Description type | Zwitterionic | |||
| Functional properties | Ideal for the study of intact membrane protein structure and non-covalent interactions. | Efficient at solubilization of challenging and delicate membrane proteins. | ||
| Ideal for sensitive membrane proteins, minimizes potential cytotoxic effects such as denaturation and aggregation. | Optimal for wide range of membrane protein types by maintaining native conformation and functionality of proteins. | Increased hydrophobicity results in enhanced solubilization and stabilization of larger hydrophobic regions or transmembrane domains. | ||
| Dialyzable | Yes | Yes | Yes | Yes |
| Aggregation number* | ~76 | Unknown | ~54 | ~131 |
| MW | 229.4 | 529.2 | 351.5 | 393.5 |
| CMC % w/v (mM) | 0.023% (~1-2) | -- (~2.9) | 0.047% (1.5) | 0.0027% (0.07) |
| Compatible with Detergent Removal Resin | Yes | Yes | Yes | No |
| Available size(s) | 1 g 5 g | 1 g 5 g | 1 g 5 g | 1 g 5 g |
| See all fos-choline detergents | ||||
* Aggregation number is the number of molecules per micelle
Not worried about maintaining protein function? See our detergents for protein solubilization
Polymers for detergent-free membrane protein isolation
Polymers represents a cutting-edge approach to membrane protein extraction, offering a detergent-free method for isolating membrane proteins along with their surrounding lipid bilayer. This innovative technology provides researchers with a powerful tool for studying membrane proteins in their native lipid environment, potentially leading to more accurate structural and functional analyses.
Like detergents, it is important to understand polymer characteristics in order to determine the best polymer for membrane protein target and intended experimental goal. A recent publication highlights the importance of selecting a polymer that effectively interacts with the specific lipid environment to maintain protein stability and functionality (1).
Characteristics of membrane protein extraction polymers
| Attribute | Unit | SMALP® 200 | SMALP® 300 |
|---|---|---|---|
| Polymer styrene-to-maleic anhydride ratio | n:m | 2:1 | 3:1 |
| Molecular weight | Da | 6,500 | 10,000 |
| Dry solids content | % w/w | 20 | 20 |
| Solvent | | Water | Water |
| pH | | 7.5 | 8.0 |
| Absolute viscosity | mPaꞏs | ≤ 5,000 | ≤ 5,000 |
| See all SMALP 200 polymers | See all SMALP 300 polymers | ||
Ordering information
See all: Protein extraction buffers, reagents, and kits
See also: Cell lysis detergents
Citations
Discover protein preparation tools
Protein sample preparation technical handbook
The Protein Preparation Handbook provides useful information on our broad selection of reagents and tools for protein extraction, clean-up, immunoprecipitation, and purification. Practical information, selection guides, and relevant data are included to help you improve your protein yield and downstream analysis.
Protein sample preparation eLearning course
This modular, animated, and narrated eLearning course was developed to provide a succinct, contextual summary of common laboratory methods and techniques required to achieve optimal results in protein assays and experiments. There are knowledge checks throughout the course to test what you have learned.
Related products and downstream applications
Resources and support
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For Research Use Only. Not for use in diagnostic procedures.