Detergent solubilization of cells

B.3 Detergent solubilization of cells

Ionic detergents such as sodium dodecyl sulphate (SDS, anionic detergent) or acetyltrimethyl ammonium bromide (cationic detergent) are very efficient at solubilizing cells but, except at low concentrations (for SDS < 0.1%) tend to disrupt proteins by destroying their secondary, ter- tiary or quaternary structure. Non-ionic detergents, such as Nonidet P-40, Triton X-100 or Renex

30, tend to be less efficient solubilizers but do preserve protein structure and protein–protein interaction. Many multichain cell-surface macromolecules have their non-covalent interchain binding preserved when cells are solubilized in detergent excess and lipids and membrane proteins trans- fer from the membrane into the detergent micelles. The configuration of most of the protein molecules and the external orientation of many is sufficiently preserved that antibodies are still able to react with antigens from solubilized cells for radioimmunoassay, immunoprecipitation, etc.

A P P E N D I X B: Basic techniques and useful data

Non-ionic detergents are able to solubilize the surface membranes of cells but leave the nuclear membrane intact; the intact nuclei are removed by centrifugation. Consequently, it is possible to solubilize cells without the viscosity changes due to released DNA. For most purposes, the aliphatic polyoxyethylene isoalcohol Renex 30 is preferable to either Nonidet P-40 or Triton X-100 (aromatic detergents of the polyoxyethylene p-t-octyl phenol series) as it does not absorb at 280 nm (and so may be used for application where UV monitoring of protein content is required), does not interfere with the Lowry estimation of protein and is not labelled during the iodination procedures described above.

MATERIALS AND EQUIPMENT Radiolabelled viable cells (10 7 lymphocytes, use pro rata and adjust for significant variations in cell surface area) Phosphate-buffered saline (PBS), containing 5 × 10 –7 M potassium iodide Renex 30, 1% v/v in PBS Protease inhibitors High-speed centrifuge, Sorval

METHOD

1 Wash the cells three times in PBS by centrifugation (150 g for 10 min at 4°C) and resuspend the dry pellet by vortexing.

2 Add 100 µl of Renex solution and mix by vigorous vortexing while adding the protease inhibitors.

3 Leave solubilized cells for 20 min on ice, vortex and then centrifuge at 250 g for 10 min at 4°C to remove the intact nuclei (and unsolubilized cell membranes, if the detergent was not in excess).

4 Clarify the solubilized membranes by centrifugation at 100 000 g for 20 min at 4°C.

5 Determine the fraction of total radioactivity associated with protein and also the specific activity, if required.

6 Store at –80°C until use. The low temperature is preferable to retard proteolytic degradation. TECHNICAL NOTES

• During cell-surface iodination, up to 10% of the apparent coupling of 125

I is through non-

covalent interaction with membrane lipids. A significant proportion of this unwanted label can

be exchanged back into the medium during the 20-min incubation on ice. • If a gel forms during solubilization the nuclei have been disrupted, usually because one of the

solutions is not isotonic. Ideally start again. However, if the cells are very valuable vortex very violently to try to reduce the viscosity by shearing the DNA and then add 40 µg/ml DNAse II

and 20 µg/ml phosphodiesterase (both final concentrations) per 10 7 cells and incubate at room

temperature for 30 min. Some proteolytic degradation is inevitable as most nucleases are con- taminated with proteinases.

• Some cells, particularly exotic protozoan parasites, tend to contain membrane-partitioned pro-

teolytic enzymes which can significantly degrade proteins from solubilized cells. In the case of Trypanosoma cruzi epimastigotes, the released proteinases can produce a Cleveland peptide map without the need to add V8 proteinase. Consequently, it is advisable to use a good range of proteinase inhibitors, add them with the aid of an assistant while vortexing the cell–detergent

A P P E N D I X B: Basic techniques and useful data 363 A P P E N D I X B: Basic techniques and useful data 363

• The final protein concentration may be measured by the Lowry technique as Renex 30, unlike Nonidet P-40 and Triton X-100, does not induce precipitation with the Folin–phenol reagent. • Incorporated radioactivity may be measured; however, lipids, which still might be carrying unexchanged 125

I, are precipitated by treatment with trichloroacetic acid (TCA). It is good prac- tice at the beginning of a series of labelling experiments to determine the amount of residual, lipid-bound radioactivity by soaking an additional TCA/ethanol-treated filter in 2 ml of a 2 : 1 mixture of chloroform–methanol for 10 min at room temperature prior to drying.

• Significant losses can occur during washing of the cell pellet. If the cells are destined for immunoprecipitation (see Section 3.13), it is acceptable to reduce the number of washes because of the rigorous washing procedure after the adsorption of the immune complex on to the immunoabsorbent. Even so, the protein-associated radioactivity should be > 60% for iodinated cells and > 85% for metabolically labelled cells.

• Renex 30 is often semisolid at room temperature; if so, warm in a 60°C water bath prior to use.