Cell lysis & protein extraction
Choosing a detergent for what the lysate is for, rather than taking whichever bottle is on the shelf. The head group decides how much is extracted and how much survives: non-ionic detergents preserve complexes and recover roughly half of a plasma-membrane protein, while ionic ones recover almost all of it and take the complexes apart.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Detergent lysis
Choosing a detergent for what the lysate is for, rather than taking whichever bottle is on the shelf. The head group decides how much is extracted and how much survives: non-ionic detergents preserve complexes and recover roughly half of a plasma-membrane protein, while ionic ones recover almost all of it and take the complexes apart.
How it differs from the standard method
- Digitonin at low concentration permeabilises the plasma membrane selectively because it binds cholesterol, which the organelle membranes have much less of.
- Triton X-100 and NP-40 absorb strongly in the ultraviolet, so a lysate made in either cannot be quantified by A280.
- CHAPS and the maltosides are zwitterionic or non-ionic alternatives used when a protein is unstable in Triton; detergent choice is often screened rather than reasoned.
- Detergent above the CMC is required, and dilution of a lysate below it can precipitate the protein it was keeping in solution.
1Lysis is a one-way door
An intact cell keeps its proteases shut inside lysosomes, its phosphatases separated from their substrates by membranes, and its proteins at the concentrations and in the complexes that make them work. Lysis destroys all of that in a second.
2Detergent strength decides which compartments open
Detergents work by replacing the lipid around a membrane protein with a micelle of their own. How far they go depends on whether they are ionic.
Hypotonic buffer
Swelling plus shear releases the cytosol and leaves membranes and nuclei behind. The starting point for fractionation, useless for a membrane protein.
0.5–1% NP-40 or Triton X-100
Roughly half of a plasma-membrane protein, complexes largely intact. The buffer for a co-IP or an activity assay.
RIPA
NP-40 plus deoxycholate plus 0.1% SDS. Near-complete extraction including nuclear and mitochondrial protein; complexes mostly gone.
2% SDS, 95 °C
Everything, unfolded. Kills proteases and phosphatases on contact, so it preserves labile modifications, but the lysate is viscous with chromatin and cannot be used for immunoprecipitation or protein assays that detergent interferes with.
3Why inhibitors go into the buffer you are about to use
Protease inhibitor cocktails are mixtures of small molecules and peptides (AEBSF or PMSF against serine proteases, E-64 against cysteine proteases, pepstatin against aspartic proteases, bestatin and leupeptin against the aminopeptidases) and several of them hydrolyse in water over hours to days. A bottle of lysis buffer that had a tablet dropped into it three weeks ago is lysis buffer with very little inhibitor in it.
4Mechanical lysis is an energy budget with a heat problem
Bacteria, yeast and plant tissue have walls that no detergent will open, so the energy has to come from somewhere else: a probe sonicator, a French press or microfluidiser, bead beating, or a freeze–thaw and grind. All of them convert most of the energy you put in into heat.
5Clearing the lysate, and the pellet you should not throw away
A crude lysate contains nuclei, unbroken cells, cytoskeletal material and sheets of membrane, all of which make loading inconsistent and columns block. Ten minutes at 10,000–16,000 × g at 4 °C removes them.
Key terms
Practise these as flashcards →The detergent concentration above which micelles form. Only above the CMC can a detergent solubilise membrane protein, which is why dilution of a lysate can precipitate what it was holding in solution.
A detergent with an uncharged head group, such as NP-40, Triton X-100 or digitonin. It dissolves lipid without competing with the charge and hydrophobic interactions that hold complexes together.
A mixed-detergent lysis buffer (typically 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in Tris-buffered saline) that extracts nearly all cell protein but disassembles most complexes.
The mass of detergent available per mass of protein and lipid being solubilised. Below roughly 3:1 the extraction is limited by detergent rather than by time.
A mixture covering the serine, cysteine, aspartic and metalloprotease classes. Several components hydrolyse in aqueous buffer within days, so it is added fresh to the volume being used.
Vanadate for tyrosine phosphatases, fluoride, β-glycerophosphate or okadaic acid for serine/threonine phosphatases. Protease cocktails do not substitute for them.
What is left after a low-speed spin that removes nuclei and unbroken cells but keeps membranes and organelles in suspension.
A dense aggregate of misfolded recombinant protein inside a bacterial cell. It pellets with the debris, so it looks like failed expression until the pellet is run on a gel.
The fraction of a sonication run during which the probe is on. A low duty cycle delivers the same total energy with a much lower peak temperature.
When to use it
- Preparing a whole-cell lysate for SDS-PAGE and a western blot, where complete extraction matters and complexes do not.
- Making a native lysate for an immunoprecipitation, a pull-down or an enzyme activity assay, where the complex or the fold has to survive.
- Releasing a recombinant protein from bacteria or yeast, where the wall has to be broken mechanically or enzymatically before anything else can happen.
- Extracting a labile modification (a phosphorylation, an acetylation, a ubiquitin chain) where the enzymes that remove it have to be inhibited or denatured within seconds of lysis.
Compared with alternatives
Prefer it when the question is where in the cell the protein is, rather than how much of it there is in total.
The step that always follows lysis, because equal volumes of two lysates are not equal amounts of protein.
The downstream application that dictates a mild non-ionic buffer; a RIPA lysate will not give you an interaction.
Use it after lysis when the sample has to be concentrated or taken out of a detergent that the next step cannot tolerate.
The usual readout, and the one whose interpretation depends entirely on whether the lanes were normalised to protein.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Choosing a detergent for what the lysate is for, rather than taking whichever bottle is on the shelf. The head group decides how much is extracted and how much survives: non-ionic detergents preserve complexes and recover roughly half of a plasma-membrane protein, while ionic ones recover almost all of it and take the complexes apart.
- Digitonin at low concentration permeabilises the plasma membrane selectively because it binds cholesterol, which the organelle membranes have much less of.
- Triton X-100 and NP-40 absorb strongly in the ultraviolet, so a lysate made in either cannot be quantified by A280.
- CHAPS and the maltosides are zwitterionic or non-ionic alternatives used when a protein is unstable in Triton; detergent choice is often screened rather than reasoned.
- Detergent above the CMC is required, and dilution of a lysate below it can precipitate the protein it was keeping in solution.