Have you ever bought a kit from a biotechnology company and followed the instructions… only to realize you’re not even sure why you’re doing certain steps in the prescribed way?
I usually use a proprietary kit to perform nuclear extraction. A quick search on google for “nuclear extraction” will yield a plethora of kits for this task. Unsurprisingly, many kits don’t provide information on the composition of their reagents. As a result, many scientists are blindly following kit instructions without knowing why certain steps are performed.
Nuclear Extraction? What’s That?
So, what exactly is nuclear extraction? Nuclear extraction is the process of separating the nuclear and cytoplasmic fractions of a cell. This procedure is used instead of whole cell lysis protocols (such as those using RIPA buffer), because whole cell lysis simply blasts the entire cell resulting in a mixture of cytoplasm and nucleus. Nuclear extraction can be useful for studying molecules that specifically interact with the nucleus, such as transcription factors which bind DNA.
In the good old days before commercialized kits, nuclear extraction was performed by whipping together a few standard lab ingredients. Of course, the procedure can still be performed this way (just hit google again and some home recipes should come up!). This article will hopefully help you to understand the rationale behind the steps in nuclear extraction.
A Step-by-Step Guide
Firstly, cells are harvested by trypsinizing or scraping and then rinsed with PBS (the way you would normally harvest cells for whole cell lysis). As with all cell extraction protocols, you need to perform extractions on ice and with protease and phosphatase inhibitors.
- Suspend the cell pellet in a hypotonic buffer. This bursts the cell wall, but keeps the nuclear membrane intact (see Cytoplasmic Extraction Buffer in the table below).
- Add detergent (such as NP40) and vortex to separate the nuclei from the cytoplasmic fraction. SDS is not recommended as it is denaturing, and the extracted proteins will not be in their native form.
- Centrifuge the solution and collect the supernatant (which should contain the cytoplasm).
- Resuspend the pellet (containing nuclei) in a nuclear extraction buffer (see Nuclear Extraction Buffer table below) that bursts the nuclear membrane. Glycerol is often included in this buffer as it helps to preserve the frozen extracts for functional experiments (such as electrophoretic mobility shift assays that assesses the ability of the nuclear protein to bind to DNA)
- Centrifuge at high speed and collect the supernatant (nuclear fraction). The final pellet should, theoretically, just be cell membrane debris which can be discarded.
Here are examples of buffer recipes to get you started…
Cytoplasmic Extraction Buffer
10 mM HEPES Buffer
1.5 mM MgCl2 Salt, for cell lysis
10 mM KCl Salt, for cell lysis. Alternatively, KCl concentration can be increased to 60mM, and the MgCl2 omitted.
0.5 mM DTT Antioxidant
1 mM EDTA Buffer. Excluded in some recipes
0.05% NP40 Detergent. Substitutes: 0.05% Igepal or Tergitol.
Nuclear Extraction Buffer
|5 mM HEPES||Buffer. Substitute: TrisCl*|
|1.5 mM MgCl2||Salt, for cell lysis. Substitute: KCl*|
|4.6 M NaCl||Salt, for cell lysis|
|0.2 mM EDTA||Buffer. Excluded in some recipes.|
|0.5 mM DTT||Antioxidant|
|26% glycerol||Retains protein functionality during freezing|
*See recipes in the references below for recommended concentrations.
Now for Some Tips!
- In the steps that involve breaking membranes (#1 and #4), vortexing is often used to facilitate lysis. However, vortexing sometimes isn’t enough. It can help to use a fine needle (e.g. 25G) to shear your cellular material.
- In the nuclear extraction step (#4), the nucleus can be difficult to lyse. Many protocols recommend vortexing every few minutes over a total period of 30 min to an hour. In my experience, increasing the vortexing time and incubation time results in a higher nuclear protein yield.
- Don’t always stick to the reagent volumes recommended by the kits. Scaling down the volume of your nuclear extraction buffer will increase your nuclear protein concentration. This is necessary if you want to make nuclear and cytoplasmic extracts of comparable concentrations, since there is almost always less total nuclear protein than cytoplasmic protein.
Checking Your Results
Once you’ve performed your nuclear extraction, how do you know that you’ve actually got nucleus in the nuclear fraction and cytoplasm in the cytoplasmic fraction? You can determine the purity of your extracts by western blotting for markers that are known to specifically reside in each of the fractions.
Common nuclear-specific markers include histones (those molecules that DNA loves to wrap itself around) and TATA binding protein (which, as you guessed, binds to a common DNA sequence, the TATA box). Cytoplasm-specific markers include heat shock proteins (which are normally found in mitochondria and should not be present in nuclear fractions) and vimentin (a cytoskeleton component).
Hopefully this article will remind you to think about the rationale behind your scientific techniques, good luck with your nuclear extractions! Have you used nuclear extraction in your work? Share your tips in the comments below!
Buffer recipes given in the table were obtained from the Abcam Nuclear Fractionation Protocol
Alternative recipes can also be found from Rockland
Abmayr SM, Yao T, Parmely T, Workman JL. Preparation of nuclear and cytoplasmic extracts from mammalian cells. Curr Protoc Mol Biol. 2006; Chapter 12: Unit 12 1.