Biological Detergents

Inevitable for the Isolation and Separation of Proteins

Membrane Protein Isolation

The isolation of membrane proteins from cell lysates or homogenates is an important biochemical operation. The process is rather delicate especially with respect to the following concerns: 1.In the process of isolation the protein has to be brought into stable solution in order to allow other cell components to be removed by centrifugation. 2.For most applications it is important that biological activity of the membrane proteins is preserved. 3.The detergent must be easily separable from the protein as well. In aqueous solutions detergents form spherical micelles which have the ability to encapsulate hydrophobic particles such as membrane proteins. The size and affinity of micelles to guest particles is highly dependent upon their composition. The formation of micelles is an equilibrium process that requires time. The detergent dissolves membrane fragments to form mixed micelles made from detergent and phospholipids. Other polymeric cell components can be removed after centrifugation. The formation of stable mixed micelles is important in the removal of membrane lipids. The preservation of biological activity represents a particular challenge. Often detergents will denature proteins at elevated concentrations. At the same time a lower concentration will reduce protein yield. Development of an isolation protocol is a highly empirical process that requires experience and extensive experimentation. Finally, the detergent must be separated from the protein. This can be achieved by ion exchange chromatography (if neutral detergents are used) or by dialysis. Biological detergents consist of an elongated molecular structure with one hydrophilic part and another consisting of a lipophilic structure (amphiphilic). Just like with regular soap, many detergents carry a charge on their hydrophilic end. However, there are also neutral detergents with no charge. Detergents can be divided into three groups based on the nature of their hyrophilic structure: ionic detergents, zwitterionic detergents and neutral detergents.

Ionic detergents carry a charge, in most cases a negative charge. These detergents include sulfonic acid esters of fatty acids or other long chain alcohols, bile acid salts or sulfonic acid derivatives of bile acids as well as phospholipids of various kind.

Selection of Biosynth's Ionic Detergents
Cat. No. - Product Name
C-2940 - Chenodeoxycholic acid, sodium salt
C-5910 - Cholic acid, sodium salt
G-5300 - Glycocholic acid, sodium salt hydrate
G-5430 - Glycodeoxycholic acid, sodium salt
S-6500 - Sodium taurocholate
S-6560 - Sodium taurodeoxycholate


Zwitterionic detergents carry both a positive and a negative charge. Examples include long chained phosphatidyl or sulfonyl choline or derivatives of zwitterionic amino acids.

Selection of Biosynth's Zwitterionic Detergents
Cat.No. - Product Name
C-5914 - Choline hexadecyl phosphate
D-6048 - 1,2-Dipalmitoyl-rac-glycero-3-phosphocholine
L-1470 - Lauroyl-DL-carnitine chloride
P-1105 - Palmitoyl-L-carnitine chloride

Neutral (non-ionic) detergents carry no charge and typically consist of sugar glycosides with long chained aglycons.

detergent-solubilize-protein

Selection of Biosynth's Neutral Detergents
Cat. No. - Product Name
D-0290 - n-Decyl-beta-D-maltopyranoside
D-3100 - Digitonin USP24
D-8820 - n-Dodecyl-beta-D-glucopyranoside
H-1340 - HECAMEG
H-1750 - n-Heptyl-beta-D-glucopyranoside
O-2010 - n-Octyl-beta-D-glucopyranoside
O-2700 - n-Octyl-beta-D-thiogalactopyranoside


Selection of the right detergent for an isolation process is highly empirical, and critical at the same time. The decision is guided by the three main concerns listed above. For example, if a protein can be purified by ion exchange chromatography the use of neutral detergents is advisable. On the other hand preservation of biological activity may require the use of bile acid salts.

Protein Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate (SDS) is an important reagent in polyacrylamide gel electrophoresis (SDS-PAGE). SDS binds to proteins yielding SDS-protein complexes with a typical mass ratio between 1.4 :1 of SDS to protein. In the process of complexation proteins dissociate and unfold such that only the primary structure is maintained and protein molecule is assuming a rod like shape. In addition the SDS sulfates double negative charge is overwhelming any intrinsic charge a protein may carry. Hence charge of the SDS protein complex is roughly proportional to molecular. Because of their defined nature SDS-Protein complexes are ideally suited for separation of proteins.

SDS is often used in combination with 1,4-Dithiothreitol (DTT) to reduce sulfide bridges prior to electophoresis. In order to prevent re-oxidation of reduced proteins the sulfides are often ireversibly alkylated with Iodoacetamide.

In some cases such as in the isolation of serum and urinary proteins the disulphide bridges are maintained and no reducing agent is used.


Selection of Biosynth's Products
Cat. No. - Product Name
S-4600 - Sodium dodecyl sulfate
D-8200 - 1,4-Dithiothreitol
D-8220 - L-1,4-Dithiothreitol


Literature:
T. Manabe, H. Oota, J. Mukai, Electrophoresis 19 (1998) 2308-2316
Ganzler K, Greve KS, Cohen AS, Karger BL, Guttman A, Cooke NC.Anal Chem. 1992 Nov 15;64(22):2665-71. High-performance capillary electrophoresis of SDS-protein complexes using UV-transparent polymer networks.

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