For soft tissue homegenization like the liver, isotonic or iso-osmotic solution (role of sucrose is to prevent osmotic choc) with buffer (important!) (low concentrations such as Tris, Hepes, at a pH between 7) is used. To prevent protein hydrolyzis, protease inhibitors (like PMSF), chelating agents (EDTA) and reducing agents (DTT) have to be added to the homogenizing solution.

In conclusion, you are right, buffering is essential for homogenizing liver tissue.

At 0.25 M, sucrose iso-osmotic. Therefore there isnt much damge to sub-cellular fractions. You can include buffers like HEPES in your sucrose medium at low concentrations such as 25-50mM.

Hope it answers your question

Unless specified, you can prepare your homogenates in phosphate buffer 100 mM pH 7.4.

But lot of enzymes need specific buffer system. G6Pase is microsomal and hence we use sucrose containing small amouts of EDTA. Glucokinase is a very sensitive enzyme therefore u need to have reducing agents like mercaptoethanol or N-acetylcysteine in the homogenization medium.

When in doubt, my suggestion is refer the original paper and try what they suggest.

Its a bad idea to look for method details in a paper that cites original method. Always read the ORIGINAL paper and adopt the protocol for ur studies.

Good luck.

Of course, if you are assaying either G6Pase or Glycogen Phosphorylase, never use phosphate buffer system because the phosphate from buffer will phenomenally increase your background readings when you do inorganic phosphate estimation.

For soft tissue homegenization like the liver, isotonic or iso-osmotic solution (role of sucrose is to prevent osmotic choc) with buffer (important!) (low concentrations such as Tris, Hepes, at a pH between 7) is used. To prevent protein hydrolyzis, protease inhibitors (like PMSF), chelating agents (EDTA) and reducing agents (DTT) have to be added to the homogenizing solution.

In conclusion, you are right, buffering is essential for homogenizing liver tissue.

Hanan Osman-Ponchet- thanks for the reply. You mentioned soft tissues.....in that case what about hard tissues (like kidney)? Won't it need isotonic homogenisation?

Apurva Kumar- thanks for the clarification.

Apurva Kumar- thanks you.. Your answer helped. But why sucrose? why can't glucose or fructose be used to form isotonic solution?

I am not sure but the idea is not to give any monosaccharides. Hope u got ur answer.

Regardless of their chemical properties, glucose and fructose are two cellular metabolites you can’t add them to a buffer, especially if you are going to assay for an enzymatic activity. There are others saccharides such as glycerol, sorbitol,…which serve the same purpose (stabilize cells or/and cellular organels, etc...) but one should always have in mind that whatever he puts in his buffer it should not be substrate for any activity contained in his cell system unless on purpose. For example people working with some microorganisms such as yeast use sorbitol because sucrose and glycerol could be metabolized. Hope that helps.

Thanks . that helped a lot.

Hi Sriharsha,

You can feed the rats with Aroclor 1254 or similar liver stimulant which helps in increasing the total enzyme content.

That depends on your enzyme. Does it require magnesium? If not, it's good to add EDTA to prevent protease activity. The sucrose is used to make the solution isotonic as was already said.

Glass dounce homogenizers work well. Make sure the strokes are slow to prevent foaming/denaturation. This may require 20 - 40 strokes, and the liver needs to be finely minced before douncing. There are also teflon homogenizers which can spin at a few hundred rpm; with these only a few strokes are needed. Sucrose is often added as a protein stabilizer as it limits osmotic shock during homogenization. Buffering is essential; keep in mind that there are always tradeoffs between optimal assay conditions and optimal purification/storage conditions.

Most of the important points have been mentioned yet. If you are worried about protease activity, PMSF and EDTA are not enough. Some other protease inhibitors for acid proteases or cathepsins could be needed. There are some commercial protease inhibitors cocktails available. It depends on the enzymes/proteins you are interested to study.

To answer your first question, sucrose is going to increase the osmolarity of the lysis solution (without adding NaCl, which could be inhibitory for the enzymes), so it keeps everything happy during the lysis of the cells and is supposed to also prevent mitochondria from breaking open and releasing enzymes.

The answer to your second question was touched on, in the first step of glycolysis glucose is a substrate for hexokinase which phosphorylates it into glucose-6 phosphate. G-6-P is the substrate for G6Pase in the last step of gluconeogenesis, so glucose is avoided to prevent these enzyme from being active. Also, if you want to assay the crude lysates for G6Pase, you do not want to have high levels of glucose around since that will affect your results (same for fructose). Sucrose can feed into glycolysis but has to be broken down first so G-6-P is not made as rapidly as with glucose.

The buffer you use needs to be assay dependent. What enzyme activities are you measuring? Surely there is an accepted buffer cited in the literature that everyone uses?

We do a lot of work looking at mitochondrial enzymes and a [175 mM] KCl/[2 mM] EDTA buffer works fine if you keep things as cold as possible. The same buffer works well for skeletal muscle and adipose tissue homogenates too. Beware of protease inhibitors in enzyme assay buffers- if your activity assay is colorimetric they can interfere.

If you are looking for a whole-tissue homogenate, then you need to use the appropriate salt buffered solution (and forget about the sucrose). As far as sucrose buffers go, usually sucrose gradient buffers are used when fractionating cells into cytosolic, mitochondrial and nuclear fractions (helps keep the membranes intact). But I'm assuming that if you are looking at enzyme activities you will want to bust open all of the membranes to free as much of the enzyme into solution as you can (unless you are comparing enzyme activities from different cellular compartments, then fractionating is necessary- just remember, you can only fractionate using fresh tissue).

If I'm homogenizing small sized samples I usually crush the tissue under liquid nitrogen into powder-like granules (before adding the buffer) and then give it a quick whiz with a pestle homogenizer. The most important thing is to keep your sample as cool as possible throughout the entire homogenization process so it doesn't disrupt the enzyme activities before you've measured them.

The answers have covered more or less everything except temperature control. It is advisable to have all solutions ice cold. The frictional heat generated during the action of the homogeniser can cause a temperature rise and may lead to loss of activity. Pe-cooling the buffered sucrose solution and also the pestle and mortar of the homogeniser itself will help to prevent unwanted rises in temperature

Yes low temp is very important. In fact we hold beaker with water with ice cubes in one hand and homogenizer cup in the other hand such that portion of the cup with tissue and buuffer is always immersed in ice bath while homogenizer is running.

Depending in what site of the cell your enzyme is located you can use glass homogenizer or use sonication. Colld buffer and cold working station ie abucket of ice or cold room is nevessary. Information about your enzyme must be collected before start.

can the above protocol be used for antioxidant assays too......like CAT, SOD, GSH and LPO?

i have used the sucrose lysis buffer solution for SOD assays and it worked

following is the recipe: 

0.25 M sucrose, 10 mM Tris (pH 8), 1mM EDTA (pH 8)] and sonicated on an ice bath (60W with 0.5 second intervals for 15 min).

it shall not work for all antioxidant assays. you may need to review standard protocols for different antioxidants. For example totaal glutathione levels may require you to deproteinise the tissue homogenate later with 5 sulfosalicylic acid etc