hey
i am using 2%OG and 1%DDM
what concentration do i need to use with DM?
is there a formula to calculate?
Why usualy using 1%DDM and 2% OG (what % is higher then the CMC?)
thanks
DDM (dodecylmaltoside) has a CMC of 0.15 mM and a molecular weight of 510.6. A 1% solution (1 g/100 ml = 10 g/L) has a concentration of (10 g/L) x (mole/510.6 g) = 0.0196 M = 19.6 mM. Therefore, a 1% solution is 130-fold above the CMC concentration.
nOG (n-octylglucoside) has a CMC of 20-25 mM and a molecular weight of 292.4. A 2% solution has a concentration of (20 g/L) x (mole/292.4) = 68.4 mM. Therefore, a 1% solution is 2.7- 3.4-fold above the CMC concentration.
The detergent concentration needed depends on how it is being used. A higher concentration is needed for dissolving the membranes, and the more membranes per volume of detergent solution the higher the detergent concentration needs to be. To keep a membrane protein in solution once it has been dissolved requires a lower detergent concentration than dissolving membranes, but the detergent concentration needed increases as the protein concentration increases. The detergent concentration usually has to be maintained above the CMC.
People tend to do what others have done before successfully, and the same goes for the use of detergents to purify membrane proteins. That is probably why those particular DDM and nOG concentrations are used. If you have the time and the desire, you can experiment with other detergents and concentrations to see what works best for your particular experiment. One motivation may be to save money. Since those detergents are expensive, you might find it worthwhile to see of you can get away with using lower concentrations or less expensive detergents.
Lauryldimethylamine-N-oxide 1percent zweiter ion
2%OG and 1%DDM nonionic
detergent protein purification
…
Table 1. Membrane Protein Purification Kit contents*
Component Amounts
supplied
CMC†
(%)
CMC†
(mM)
Detergent
class
His Mag Sepharose Ni 3 × 1 ml
n-Dodecyl-b-D-maltoside (DDM) 1 ml‡ 0.009 0.17 Non-ionic
n-Decyl-b-D-maltoside (DM) 1 ml‡ 0.09 1.8 Non-ionic
Lauryldimethylamine-N-oxide
(LDAO)
1 ml‡
0.02
1–2
Zwitter-ionic
n-Dodecylphosphocholine
(FOS12)
1 ml‡
0.05
1.5
Non-ionic
Dodecyl octaethyleneglycol
ether (C12E8)
1 ml‡
0.005
0.09
Non-ionic
Cyclohexyl-1-pentyl-b-Dmaltoside
(CYMAL™-5)
1 ml‡
0.12
2–5
Zwitter-ionic
n-Octyl-b-D-glucoside (OG) 1 ml§ 0.53 18–20 Non-ionic
Phosphate buffer (stock) pH 7.4
(160 mM sodium phosphate,
4 M NaCl) 100 ml
2 M imidazole pH 7.4 100 ml
* All detergents are from Anatrace™ Maumee, Ohio, USA (Affymetrix)
Table 2. Detergents and concentrations used in screening
Detergents Solubilization Purification
DDM 1% 0.1%
DM 1% 0.2%
LDAO 1% 0.2%
FOS12 1% 0.1%
C12E8 1% 0.1%
CYMAL-5 1% 0.2%
OG 2% 1%
DDM (dodecylmaltoside) has a CMC of 0.15 mM and a molecular weight of 510.6. A 1% solution (1 g/100 ml = 10 g/L) has a concentration of (10 g/L) x (mole/510.6 g) = 0.0196 M = 19.6 mM. Therefore, a 1% solution is 130-fold above the CMC concentration.
nOG (n-octylglucoside) has a CMC of 20-25 mM and a molecular weight of 292.4. A 2% solution has a concentration of (20 g/L) x (mole/292.4) = 68.4 mM. Therefore, a 1% solution is 2.7- 3.4-fold above the CMC concentration.
The detergent concentration needed depends on how it is being used. A higher concentration is needed for dissolving the membranes, and the more membranes per volume of detergent solution the higher the detergent concentration needs to be. To keep a membrane protein in solution once it has been dissolved requires a lower detergent concentration than dissolving membranes, but the detergent concentration needed increases as the protein concentration increases. The detergent concentration usually has to be maintained above the CMC.
People tend to do what others have done before successfully, and the same goes for the use of detergents to purify membrane proteins. That is probably why those particular DDM and nOG concentrations are used. If you have the time and the desire, you can experiment with other detergents and concentrations to see what works best for your particular experiment. One motivation may be to save money. Since those detergents are expensive, you might find it worthwhile to see of you can get away with using lower concentrations or less expensive detergents.
Probably one of the cheapest detergents used for protein purification is Triton X-100. Besides, it can be used during protein purification based on chromatographic columns (ion exchange, hydrophobic columns or even gel filtration). Concentrations around 2% (p/V) are usually used to dissolve membranes and then, this concentrations is decreased up to 0.2 % in those buffers used to perform chromatographic steps.
Triton X-100 is indeed much less expensive that DDM or nOG, but you must be careful about how the Triton is stored, because it oxidizes in air to form peroxides that can damage your protein. Store it under inert gas, or frozen. For convenience, you can make a 10% solution and store it frozen in aliquots. You can also buy it as a 10% solution in 10-ml ampules under inert gas from Thermo-Fisher, but that is obviously much more expensive.
Thank you all for your help
Thank you especially for adding calculation method
You helped me a lot
- Badges
- Science method