10. The ClearNative gel electrophoresis works without using an
anionic dye. Therefore, this method can be used for the separation of membrane proteins prepared with an anionic detergent. For nonionic or zwitterionic detergents, the suitable
method is the BlueNative gel electrophoresis, which uses a
negatively charged Coomassie Brilliant Blue G250 dye that
forms a complex with the protein to enable the gel migration.
11. The choice for the method of protein detection depends on the
amount of protein loaded to the gel. The immunodetection by
Western blot is more sensitive than the Coomassie staining but
requires more steps.
12. For size exclusion chromatography, it is recommended to
degas and filter all solutions through a 0.22 μm filter. However,
some detergents may interact with the filter membrane and be
retained. If in doubt, do not filter the eluent buffer.
13. For size exclusion chromatography, it is recommended to
choose the optimal fractionation range of the column to ensure
better separation between aggregates and non-aggregates for
high molecular proteins.
References
1. Almen MS et al (2009) Mapping the human
membrane proteome: a majority of the human
membrane proteins can be classified according
to function and evolutionary origin. BMC Biol
7:50
2. Overington JP, Al-Lazikani B, Hopkins AL
(2006) How many drug targets are there? Nat
Rev Drug Discov 5(12):993–996
3. Seddon AM, Curnow P, Booth PJ (2004)
Membrane proteins, lipids and detergents:
not just a soap opera. Biochim Biophys Acta
1666(1–2):105–117
4. Duquesne K, Sturgis JN (2010) Membrane
protein solubilization. Methods Mol Biol
601:205–217
5. Eshaghi S (2009) High-throughput expression
and detergent screening of integral membrane
proteins. Methods Mol Biol 498:265–271
6. Agez M et al (2017) Molecular architecture of
potassium chloride co-transporter KCC2. Sci
Rep 7(1):16452
7. Desuzinges Mandon E et al (2017) Novel systematic detergent screening method for membrane proteins solubilization. Anal Biochem
517:40–49
8. Ashok Y, Nanekar R, Jaakola VP (2015) Defining thermostability of membrane proteins by
western blotting. Protein Eng Des Sel 28
(12):539–542
9. Igonet S et al (2018) Enabling STD-NMR
fragment screening using stabilized native
GPCR: a case study of adenosine receptor. Sci
Rep 8(1):8142
10. Nguyen KA et al (2018) Glycosyl-substituted
Dicarboxylates as detergents for the extraction,
overstabilization, and crystallization of membrane proteins. Angew Chem Int Ed Engl 57
(11):2948–2952
11. Matar-Merheb R et al (2011) Structuring
detergents for extracting and stabilizing functional membrane proteins. PLoS One 6(3):
e18036
12. Hardy D et al (2018) The yin and yang of
solubilization and stabilization for wild-type
and full-length membrane protein. Methods
147:118–125
13. Desuzinges Mandon E et al (2017) Expression
and purification of native and functional influenza a virus matrix 2 proton selective ion channel. Protein Expr Purif 131:42–50
14. Jawhari A et al (2006) Structure and oligomeric state of human transcription factor
TFIIE. EMBO Rep 7(5):500–505
Solubilization and Stabilization of Membrane Proteins
267
anionic dye. Therefore, this method can be used for the separation of membrane proteins prepared with an anionic detergent. For nonionic or zwitterionic detergents, the suitable
method is the BlueNative gel electrophoresis, which uses a
negatively charged Coomassie Brilliant Blue G250 dye that
forms a complex with the protein to enable the gel migration.
11. The choice for the method of protein detection depends on the
amount of protein loaded to the gel. The immunodetection by
Western blot is more sensitive than the Coomassie staining but
requires more steps.
12. For size exclusion chromatography, it is recommended to
degas and filter all solutions through a 0.22 μm filter. However,
some detergents may interact with the filter membrane and be
retained. If in doubt, do not filter the eluent buffer.
13. For size exclusion chromatography, it is recommended to
choose the optimal fractionation range of the column to ensure
better separation between aggregates and non-aggregates for
high molecular proteins.
References
1. Almen MS et al (2009) Mapping the human
membrane proteome: a majority of the human
membrane proteins can be classified according
to function and evolutionary origin. BMC Biol
7:50
2. Overington JP, Al-Lazikani B, Hopkins AL
(2006) How many drug targets are there? Nat
Rev Drug Discov 5(12):993–996
3. Seddon AM, Curnow P, Booth PJ (2004)
Membrane proteins, lipids and detergents:
not just a soap opera. Biochim Biophys Acta
1666(1–2):105–117
4. Duquesne K, Sturgis JN (2010) Membrane
protein solubilization. Methods Mol Biol
601:205–217
5. Eshaghi S (2009) High-throughput expression
and detergent screening of integral membrane
proteins. Methods Mol Biol 498:265–271
6. Agez M et al (2017) Molecular architecture of
potassium chloride co-transporter KCC2. Sci
Rep 7(1):16452
7. Desuzinges Mandon E et al (2017) Novel systematic detergent screening method for membrane proteins solubilization. Anal Biochem
517:40–49
8. Ashok Y, Nanekar R, Jaakola VP (2015) Defining thermostability of membrane proteins by
western blotting. Protein Eng Des Sel 28
(12):539–542
9. Igonet S et al (2018) Enabling STD-NMR
fragment screening using stabilized native
GPCR: a case study of adenosine receptor. Sci
Rep 8(1):8142
10. Nguyen KA et al (2018) Glycosyl-substituted
Dicarboxylates as detergents for the extraction,
overstabilization, and crystallization of membrane proteins. Angew Chem Int Ed Engl 57
(11):2948–2952
11. Matar-Merheb R et al (2011) Structuring
detergents for extracting and stabilizing functional membrane proteins. PLoS One 6(3):
e18036
12. Hardy D et al (2018) The yin and yang of
solubilization and stabilization for wild-type
and full-length membrane protein. Methods
147:118–125
13. Desuzinges Mandon E et al (2017) Expression
and purification of native and functional influenza a virus matrix 2 proton selective ion channel. Protein Expr Purif 131:42–50
14. Jawhari A et al (2006) Structure and oligomeric state of human transcription factor
TFIIE. EMBO Rep 7(5):500–505
Solubilization and Stabilization of Membrane Proteins
267