Compositions and methods for membrane protein detergent stability screen
The invention provides methods to assess protein stability and to obtain sizing information. In one aspect, the screen comprises a 94 detergent panel and a series of MWCO filtered microplates. A protein of interest is bound to an affinity matrix and aliquoted into a 96-well microplate. Wells containing the immobilized protein are washed in the new detergent and then eluted in the new detergent into a collection plate. Protein not stable in the new detergent is precipitated on the resin and not present in the elutions. Half of the elution is passed through a high (i.e., 300 kDa) MWCO microplate and the other half through a low (i.e., 100 kDa) MWCO microplate. Elutions from the microplates are spotted on a nitrocellulose membrane, visualized by Western analysis (or by some other method), and quantified. The high MWCO provides stability readout and the ratio of low/high kDa provides sizing information.
1. A method for determining the stability and size of a protein in at least one detergent using a rapid differential filtration detergent screening assay, said method comprising:
a) obtaining a solution comprising said protein in a first detergent;
b) adding an effective amount of an affinity resin to said solution;
c) adding an aliquot of said solution comprising said protein in a first detergent and an affinity resin to a first chamber, said first chamber comprising a filter wherein said pore size is about 0.2 μm;
d) washing said first chamber with a wash solution comprising a different detergent wherein the concentration of said different detergent is the critical micelle concentration of said different detergent;
e) eluting said protein in said first chamber with an elution solution comprising said different detergent and collecting the eluate;
f) passing an aliquot of said eluate through a second chamber comprising a high molecular weight cut-off filter and another aliquot of said eluate through a third chamber comprising a low molecular weight cut-off filter;
g) measuring the amount of protein in the eluate passing through the high molecular weight cut-off filter and the amount of protein in the eluate passing through the low molecular weight cut-off filter; and
h) comparing the amount of protein eluted through the high molecular weight cut-off filter with the amount of protein eluted through the low molecular weight cut-off filter, thereby determining the stability and size of a protein in at least one detergent using a rapid differential filtration detergent screening assay.
2. The method of claim 1 , wherein said first chamber is a well of a multiwell microplate.
3. The method of claim 1 , wherein said second chamber is a well of a multiwell microplate, further wherein each well comprises a high molecular weight cut-off filter.
4. The method of claim 1 , wherein said third chamber is a well of a multiwell microplate, further wherein each well comprises a low molecular weight cut-off filter.
5. The method of claim 2 , wherein at least two wells are used.
6. The method of claim 5 , wherein the first detergent in step a) is tested with multiple different detergents in step d), wherein a different first well is used for each of said multiple different detergents tested and optionally one or more wells comprise a positive control and one or more wells comprise a negative control.
7. The method of claim 6 , wherein when multiple detergents are tested, each well comprising a detergent comprises a different detergent than the other wells comprising a detergent, and optionally one or more wells comprise a positive control and one or more wells comprise a negative control.
8. The method of claim 1 , wherein said high molecular weight cut-off filter is about 300 kDa.
9. The method of claim 1 , wherein said low molecular weight cut-off filter is about 100 kDa.
10. The method of claim 7 , wherein said multiwell plate is a 96, 384, or 1536 well plate.
11. The method of claim 10 , wherein said 96 well plate is a Society for Biomolecular Sciences format plate.
12. The method of claim 10 , wherein 94 different detergents are tested.
13. The method of claim 1 , wherein said different detergent and said different detergents concentration is selected from the following table:
[Det]
Well
Abbrev.
Name
mM
A1
A2
A3
Z3-12
n-Dodecyl-N,N-dimethyl-3-ammonio-1-
(2.8)
propanesulfonate
A4
Z3-14
-n-Tetradecyl-N,N-dimethyl-3-ammonio-
(0.2)
1-propanesulfonate
A5
DMG
n-Decyl-N,N-dimethylglycine
(19)
A6
DOMG
n-Dodecyl-N,N-dimethylglycine
(1.5)
A7
DAO
n-Decyl-N,N-dimethylamine-N-oxide
(10.5)
A8
UDAO
n-Undecyl-N,N,-dimethylamine-N-oxide
(3.2)
A9
LDAO
n-Dodecyl-N,N-dimethylamine-N-oxide
(1)
A10
C-DDFOS
Cyclododecyl-1-phosphocholine
(22)
A11
CF-4
4-Cyclohexyl-1-butylphosphocholine
(14)
A12
CF-5
5-Cyclohexyl-1-pentylphosphocholine
(4.5)
B1
CF-6
6-Cyclohexyl-1-hexyphosphocoline
(2.68)
B2
CF-7
7-Cyclohexyl-1-heptylphosphocholine
(0.62)
B3
FC-10
n-Decylphosphocholine
(11)
B4
FC-11
n-Undecylphosphocholine
(1.85)
B5
FC-12
n-Dodecylphosphocholine
(1.5)
B6
FC-13
n-Tridecylphosphocholine
(0.75)
B7
FC-14
n-Tetradecylphosphocholine
(0.12)
B8
FC-I11
2,8-Dimethyl-5-nonylphosphocholine
(26.6)
B9
FC-I11-6U
Undecyl-6-phosphocholine
(25.8)
B10
FC-I9
2,6-Dimethyl-4-heptylphosphocholine
(32)
B11
FC-U10-11
10-Undecylenyl-1-phosphocholine
(6.2)
B12
DHPC
1,2-Diheptanoyl-sn-glycero-3-
(1.4)
phosphocholine
C1
LPC-10
1-Decanoyl-2-hydroxy-sn-glycero-3-
(8)
phosphocholine
C2
LPC-12
1-Lauroyl-2-hydroxy-sn-glycero-3-
(0.7)
phosphocholine
C3
FOSFEN-9
Nonylphenylphosphocholine
(1.35)
C4
CHAPS
3-[(3-Cholamidopropyl)dimethylamminio]-
(8)
1-propanesulfonate
C5
CHAPSO
3-[(3-Cholamidopropyl)dimethylamminio]-
(8)
2-hydroxy-1-propanesulfonate
C6
DDMAU
n-Dodecyl-N,N-
(0.13)
(dimethylammonio)undecanoate
C7
DDMAB
n-Dodecyl-N,N-(dimethylammonio)butyrate
(4.3)
C8
LAPAO
3-Dpdecylamido-N,N′-dimethylpropyl
(1.6)
amine oxide
C9
TRIPAO
3-(3 Butyl-3-phenylheptanamido)-N,N-
(4.5)
dimethylpropan-1-amine oxide
C10
T-20
Polyoxyethylene(20) sorbitane monolaurate
(0.059)
C11
BRIJ-35
Polyoxyethylene lauryl ether
(0.091)
C12
TX-100
Tetramethylbutyl)phenyl]-ω-hydroxy-
(0.23)
poly(oxy-1,2-ethanediyl), average MW 647
D1
TX-114
Teteamethylbutyl)phenyl]-ω-hydroxy-
(0.2)
poly(oxy-1,2-ethanediyl), average MW 536
D2
TX-305
Teteamethylbutyl)phenyl]-ω-hydroxy-
(0.65)
poly(oxy-1,2-ethanediyl),
average MW 1526
D3
TX-405
Teteamethylbutyl)phenyl]-ω-hydroxy-
(0.81)
poly(oxy-1,2-ethanediyl),
average MW 1967
D4
NID-P40
[Octylphenoxy]polyethoxyethanol
(0.3)
D5
APO8
Dimethyloctylphosphine oxide
(40)
D6
APO9
Dimethylnonylphosphine oxide
(10)
D7
APO10
Dimethyldecylphosphine oxide
(4.66)
D8
APO11
Dimethylundecylphosphine oxide
(1.2)
D9
APO12
Dimethyldodecylphosphine oxide
(0.57)
D10
C6E3
Triethylene glycol monohexyl ether
(23)
D11
C6E4
Tetraethylene glycol monohexyl ether
(30)
D12
C6E5
Pentaethylene glycol monohexyl ether
(37)
E1
C7E5
Pentaethylene glycol monoheptyl ether
(21)
E2
C8E4
Tetraethylene glycol monooctyl ether
(8)
E3
C8E5
Pentaethylene glycol monooctyl ether
(7.1)
E4
C8E6
Hexaethylene glycol monooctyl ether
(10)
E5
C10E5
Pentaethylene glycol monodecyl ether
(0.81)
E6
C10E6
Hexaethylene glycol monodecyl ether
(0.9)
E7
C10E9
Polyoxyethylene(9)decyl ether
(1.3)
E8
C12E8
Octaethylene glycol monododecyl ether
(0.09)
E9
C12E9
Polyoxyethylene(9)dodecyl ether
(0.05)
E10
C12E10
Polyoxyethylene(10)dodecyl ether
(0.2)
E11
C13E8
Polyoxyethylene(8)tridecyl ether
(0.1)
E12
CHAP
N,N′-bis-(3-D-
(2.9)
Gluconamidopropyl)cholamide
F1
CHAP-D
N,N′-bis-(3-D-
(1.4)
Gluconamidopropyl)deoxycholamide
F2
OHES
Octyl-2-hydroxyethyl-sulfoxide
(24.2)
F3
RDHPOS
Rac-2,3-dihydroxypropyloctylsulfoxide
(24.2)
F4
GX-100
Polyoxyethylene(10) Isotridecyl Ether
(0.15)
F5
HTG
n-Heptyl-β-D-thioglucopyranoside
(29)
F6
OG
n-Octyl-β-D-glucopyranoside
(18)
F7
NG
n-Nonyl-β-D-glucopyranoside
(6.5)
F8
CYGLU-3
3-Cyclohexyl-1-propyl-β-D-glucoside
(28)
F9
HECAMEG
Methyl-6-O-(N-heptylcarbamoyl)-α-D-
(19.5)
glucopyranoside
F10
HEGA-9
Nonanoyl-N-hydroxyethylglucamide
(39)
F11
C-HEGA-10
Cyclohexylbutanoyl-N-
(35)
hydroxyethylglucamide
F12
C-HEGA-11
Cyclohexylbutanoyl-N-
(11.5)
hydroxyethylglucamide
G1
CYMAL-3
3-Cyclohexyl-1-propyl-β-D-maltoside
(30)
G2
CYMAL-4
4-Cyclohexyl-1-butyl-β-D-maltoside
(7.6)
G3
CYMAL-5
5-Cyclohexyl-1-pentyl-β-D-maltoside
(2.4)
G4
CYMAL-6
6-Cyclohexyl-1-hexyl-β-D-maltoside
(0.56)
G5
CYMAL-7
7-Cyclohexyl-1-heptyl-β-D-maltoside
(0.19)
G6
DMHM
2,6-Dimethyl-4-heptyl-β-D-maltoside
(27.5)
G7
OM
n-Octyl-β-D-maltopyranoside
(19.5)
G8
NM
n-Nonyl-β-D-maltopyranoside
(6)
G9
DαM
n-Decyl-α-D-maltopyranoside
(1.6)
G10
DM
n-Decyl-β-D-maltopyranoside
(1.8)
G11
UDαM
n-Undecyl-α-D-maltopyranoside
(0.58)
G12
UDM
n-Undecyl-β-D-maltopyranoside
(0.59)
H1
ωUDM
ω-Undecylenyl-β-D-maltopyranoside
(1.2)
H2
DDαM
n-Dodecyl-α-D-maltopyranoside
(0.15)
H3
DDM
n-Dodecyl-β-D-maltopyranoside
(0.17)
H4
TDM
n-Tridecyl-β-D-maltopyranoside
(0.03)
H5
OTM
n-Octyl-β-D-thiomaltopyranoside
(8.5)
H6
NTM
n-Nonyl-β-D-thiomaltopyranoside
(3.2)
H7
DTM
n-Decyl-β-D-thiomaltopyranoside
(0.9)
H8
UDTM
n-Undecyl-β-D-thiomaltopyranoside
(0.21)
H9
DDTM
n-Dodecyl-β-D-thiomaltopyranoside
(0.05)
H10
S-8
n-Octanoyl-β-D-fructofuranosyl-α-D-
(24.4)
glucopyranoside
H11
S-10
α-D-Glucopryanoside, β-D-
(2.5)
Fructofuranosyl Monodecanoate
H12
S-12
n-Monododecanoate-α-D-
(0.3)
glucopyranoside, β-D-Fructofuranosyl.
14. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 1000 micrograms or less of said protein.
15. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 500 micrograms or less of said protein.
16. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 400 micrograms or less of said protein.
17. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 200 micrograms or less of said protein.
18. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 100 micrograms or less of said protein.
19. The method of claim 1 , wherein said solution comprising said protein in a first detergent comprises about 50 micrograms or less of said protein.
20. The method of claim 1 , wherein said method is performed in less than about two hours.
21. The method of claim 1 , wherein said method is performed in less than about 1 hour.
22. The method of claim 1 , wherein said protein amounts are determined by dot blot or Western blot analysis.
23. The method of claim 22 , wherein said protein amounts determined from the high molecular weight cut-off dot blots are normalized and plotted with the ratio of low:high normalized intensities and the values grouped into quartiles.
24. The method of claim 22 , wherein said protein amounts determined from the high molecular weight cut-off dot blots are normalized and plotted graphically on the abscissa while the ratio of low:high normalized intensities are plotted on the ordinate.
25. The method of claim 1 , further wherein said method is used to screen detergent mixtures, additives, ionic strength, and pH.
26. The method of claim 1 , wherein said protein is a membrane protein.
27. The method of claim 1 , wherein said different detergent has zwitterionic or nonionic headgroups.
28. The method of claim 1 , wherein at least one of said first, second, or third chambers is subjected to centrifugation to enhance the filtration process.