Subcellular fractionation - (Jul/20/2004 )
I'm having trouble with trying to fractionate HeLa cells. When I probe for some proteins by Western, they show up in all fractions, from 600g spin to 100000 g spin. Is this a problem with my fractionation, or does it say something about the proteins? I thought maybe my homogenization was poor. Any ideas?
Homogenization is a critical step and should be performed always at 4C. There are several protocols for fractionation in the internet but you have to try some to check which one works best for the type of cells you are using and type of cellular organelles you are interested in separate.
Gentle homogenization conditions should be applied to limit possible breakage of endosomes and lysosomes, particularly when using fluid-phase markers. Clearly, the markers should remain entrapped in vesicles (latent) after homogenization.
In addition, harsh conditions should always be avoided to limit the breakage of lysosomes and consequent proteolysis by released hydrolases.
Each step of the homogenization process should be monitored using a phase-contrast microscope to check for cellular breakup and for damage of already released nuclei. If cells grow in tightly interacting monolayers, sheets of cells which are not homogenized properly will always be present whereas already released nuclei tend to break upon over-homogenization. The result of the prep will always be a balance between efficiency (unbroken cells will be lost in the nuclear pellet) and quality of released subcellular compartments, the latter being more important.
I will paste here a file that I believe it can give you some important information about the components of the lysis buffer you should or shouldn’t use!
Techniques of cell fractionation
Fractionation proceeds in two consecutive stages:
1) Homogenization (disrupts the tissue and
releases cellular components)
2) Centrifugation (separates the individual
components according to density, size
Problems of cell fractionation
Presence of cell wall
Forces required to disrupt cell wall may rapture cell organelles.
Presence of large vacuoles
Disruption of the vacuole can release toxic materials (e.g. phenols) and hydrolytic enzymes that can destroy cell organelles.
The challenge of cell fractionation
To chose methods of cell disruption and incubation of subcellular fractions that are appropriate to the particular investigation and which minimizes artefacts.
Slightly hypo-osmotic or iso-osmotic – to preserve structural integrity
* Osmoticums: sucrose, manitol, sorbitol
* Chelating agents: EDTA or EGTA
(remove Ca2+ or Mg2+ which are required
by membrane proteases)
* Protease inhibitors
* Ca2+ or Mg2+
Role/effects of Ca2+ and Mg2+ in homogenization medium
Required for membrane marker enzyme activity
Maintain nuclear integrity
Can cause membrane aggregation
Decrease respiratory control in mitochondria
Problems caused by phenols
Can hydrogen bond with peptide bonds of proteins.
Can be transformed into quinones. Quinones can react with reactive groups of proteins to form a dark pigment, melanin. Melanin can inactivate many enzymes.
Can form ionic interactions with basic amino acids of proteins.
Can form hydrophobic bonds with hydrophobic regions of proteins.
Removal of phenols
Must be removed as soon as possible
Best removed by using adsorbents (e.g. polyvinylpyrrolidone, bovine serum albumin) that bind to the phenols or quinones.
Phenol effects can be minimized by using protective agents (e.g. borate, sulfites, mercaptobenzothiazole) that inhibit the phenol activity.
Temperature requirements for cell fractionation
Performed at 4°C to reduce the activity of proteases.
Other compounds/conditions used in homogenization media
Disulfide-reducing agents (help maintain activity of some enzymes)
pH 7.5-8.0* (cytoplasmic pH)
* reduce lipid breakdown
Things to keep in mind
All stages of cell fractionation must be performed at 4°C – to minimize protease activity.
All media and apparatus should be precooled and maintained at 4°C.
Unwanted organelle damage may be caused by shearing forces required to break the cell wall.
Enzyme activity may be lost if there is foaming of the homogenate in the high-speed blender.
Physical methods of cell disruption
Solid methods: disruption of cells results from the shearing forces generated between the cells and a solid abrasive.
Tools/materials: 1) pestle, 2) mortar, 3) abrasive material (sand, silica, alumina), 4) homogenization medium/buffer, 5) tissue.
Liquid shear methods: disruption of cells results from the shearing forces generated between the tissue and liquid medium/homogenization buffer
Liquid shear methods:
Tissue homogenizers – generate mild shearing forces.
Microhomogenizers – designed to disrupt small amounts of tissue/cells; generate low shearing forces.
Pressure homogenization – designed to disrupt cultured cells; 1) cells equilibrated with an inert gas (argon) imbibe the gas; when the cells are suddenly returned to atmospheric pressure, the newly formed gas bubles inside the cytoplasm rapture the membranes.
Osmotic shock – protoplasts ruptured in a hypoosmotic medium.
Freezing/thawing technique – ice crystals rapture the cells
Non-physical methods of cell disruption
Organic solvents (chloroform/methanol mixtures) - dissolve membrane lipids (destroy membranes) and release integral proteins and subcellular components.
Drawback: do not preserve morphological or metabolic integrity
Chaotropic anions (potassium thiocyanate, potassium bromide, lithium diiodosalicylate) – destabilize lipid membranes consequently, the subcellular components are being released.
Non-physical methods of cell disruption
Detergents – solubilize the integral membrane proteins by interacting with the phospholipid bilayer.
1) Anionic (sodium dodecyl sulfate, SDS), denaturing
2) Non-denaturing (deoxycholate), disrupts the
membrane, preserves proteins in their native state.
3) Non-ionic (Triton X-100), disrupts the membrane, do
not destroy enzyme activity of integral membrane
Non-physical methods of cell disruption
Enzymatic digestion of the cell wall – results in protoplasts, which are subsequently subjected to one of the previous methods of cell/protoplast disruption.
Mixture of enzymes is used: chitinases, pectinases, lipases, proteases, cellulases.
Note: the general protocol of cell disruption is
well established but the precise
components/concentration of homogenization
medium must be established for individual
The separation of subcellular fractions is achieved by centrifugation.
Particles of different density, size, and shape sediment at different rate in a centrifugal field.
Factors of the sedimentation rate:
* particle size and shape
* the viscosity of suspending medium
* centrifugal field
The particle remain stationary when the density of the particle and the density of the centrifugation medium are equal
Types of centrifugation
Separates particles as a function of size and density
A particular centrifugal field is chosen over a period of time
Pellet particles of larger mass
Sample distributed throughout the medium
Subjected to repeated steps
Centrifugation medium is characterized by a positive increment in density – density gradient.
Sample is applied on the top of density gradient as a narrow band.
Particles separate into a series of bands in accordance to centrifugal field, size and shape of the particle and difference in density between the particle and the suspending medium.
Mostly used to separate nucleic acids.
Based solely on the density of the particles.
Unaffected by the size or the shape of the particles.
Separation medium – density gradient medium.
Nature of density gradient materials
Inert towards the biological material, the centrifuge tubes and rotor.
No interference with the monitoring of the sample material.
Easy separation from the fraction after centrifugation.
Easy to monitor the concentration of the gradient medium.
Stable in solution and available in a pure and analytical form.
Exert minimal osmotic pressure.
Cross-contamination and enzyme markers
Cross-contamination is a common problem associated with subcellular fractionation.
Cross-contamination can be easily assessed by enzyme markers.
Each cellular fraction comprises a unique or a combination of unique enzyme activities which can be assessed.
Examples of enzyme markers
Soluble cytoplasmic proteins (i.e not associated with the cytoskeleton, or any membrane compartments) will show up in all your fractions. The rpm you are using is not enough for spinning down these proteins.
For subcellular fractionation you should try sucrose gradients after you've lysed your cells, and subsequently blot for various compartments using antibodies as specific markers.
Could you give some example for the antibody as marker of subcellular fraction?
e.g. for membrane fraction...
Follow-up to my previous post. Thank you all for helpful advice.
I've cleaned up the data a little but it's still strange. My nuclear-localized marker (PARP) comes out predominantly in the heavy membrane spin (100,000 g), while my cytoplasmic marker (tubulin) appears predominantly in the cytosolic fraction, but also considerably in the low (600 g) and medium speed (5000 g) pellets. What do these data mean?