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

• For protein purification each detergent micelle should contain at most one protein molecule, as proteins in the same micelle cannot be separated. Because the actual number of proteins per micelle is Poisson-distributed, you need 10 micelles per protein molecule. If you assume the average molecular mass of proteins to be 100 kDa, a 1 mg/ml protein solution will be 10 µM, and you need the micelle concentration to be 100 µM.
• One detergent micelle can solubilise about 10 molecules of lipid. If you assume a 1 mg/ml membrane protein solution to also contain about 1 mg/ml lipid (actually somewhat more, but this estimate is good enough for our purposes), and the average molecular mass of lipids to be 750 Da, the solution is about 1.3 mM in lipid and you need 130 µM micelles to solubilise it.
• Thus, you need the total micelle concentration to be 230 µM. To calculate the amount of detergent, you need to multiply this with the aggregation number (number of detergent molecules per micelle) \bar{m} and then add the critical micellar concentration (cmc) of the detergent.
• For example, CTAB has a cmc of 1 mM, a \bar{m} of 170 molecules per micelle and a Mr of 364.5 Da. Thus, to solubilise 1 mg/ml membrane protein, you need 40.1 mM (14.9 mg/ml) of CTAB.

This calculation uses a lot of hand waving, but predicts experiments quite accurately. You can check this result by using 0.2 ml tubes in a tabletop ultracentrifuge to spin samples at 100 000 g for 60 min, all proteins still in the supernatant are operationally considered solubilised. Use 1/4, 1/2, 1, 2 and 4 times the calculated detergent concentration and plot protein concentration of the supernatant as function of detergent concentration. If you have an assay for the activity of your protein, plot that too. The optimal detergent concentration it that which solubilises the protein efficiently without killing you protein's activity.