Detergents are used in a wide range of experiments and sometimes figuring out which one to use can be quite tricky (something we take a closer look at here). This choice can be made even more confusing if you’re unsure as to how detergents actually work!
Why permeabilise cell membranes?
In order to visualise the inner workings of cells scientists have developed an array of tools or tags (such as dyes, antibodies (personally our favourite method :-P), probes etc.) that are able to bind specifically to molecular components and give a visual read out of their presence.
However, if these components reside within a cell membrane then the tags are not able to access their target. It is here detergents come in, detergents can be used to perforate cell membranes, allowing tags to access the inside of the cell.
Three steps are usually followed to prepare a cell for intracellular staining:
- The cell membrane is stabilised or fixed by either an aldehyde (pramformadehyde, formaldehyde or glutaralaldehyde) or an organic solvent (methanol/ethanol/acetone). Aldehyde fixation increases the permeability of a cell but not by enough for efficient intracellular labelling. If fixed by an organic solvent no further permeabilisation step is required.
- The cell membrane is permeabilised using a detergent or solvent.
- The cells are incubated with the stain or tag.
- However, when a process is optimised it is possible to do all this in one step and many of the commercial kits available are set up to work like this [3,4].
When would I use a detergent?
The most frequent uses of detergents are:
- Wash buffers: low concentrations of detergents are used to reduce nonspecific binding.
- Permeabilisation: cell membranes can be perforated by detergents to enable intracellular staining.
- Membrane solubilisation: membrane components can be extracted using detergents for immunoprecipitation assays.
What is a detergent and how do they work?
See Figure one. Detergents are made up of monomers that have a hydrophilic head group and a hydrophobic tail(a). In high enough concentrations the monomers form micelles(b). When detergents interact with the lipid membrane of a cell they become embedded.
When the concentration increases the detergents will start to influence the structure of a membrane and begin to form pores(c). If the concentration continues to increase membrane components can become fully encircled by a micelle which then buds off from the membrane(d). This is how membrane components can be lost [1,2].
1. Lacaille-Dubois MA1, Wagner H. A review of the biological and pharmacological activities of saponins. Phytomedicine. 1996 Mar;2(4):363-86. doi: 10.1016/S0944-7113(96)80081-X. PMID : 23194774
– From Liz and the CiteAb team