Whenever we homogenise the tissues for preparing samples for Western blot or any other experiment (i.e. enzyme assay, etc.), a lot of frothing occurs. How can we decrease the amount of frothing? And does the frothing affect the sample quality?
Frothing can be harmful for certain enzymes, as all the foam bubbles increase the interfase between gas and aquous extract, so a lot of oxygen can enter the liquid and that will help the plant enzyme polyphenol oxidase to cause the typical browning you see when you cut apples or pears and leave the cuts exposed to the air for a few minutes. Some enzymes can be affected during these oxidations and it will reduce activity, although less problems would be expected for western blotting under denaturing conditions. In general, I try to avoid frothing as much as I can. Foaming depends on the extraction buffer (does it have detergents?), the type of tissue (does it have lots of lipids?) and the homogenisation method (do you grind with mortar and pestle, or do you have a blender?). Gentle homogenisation in a mortar on ice (with ice-cold extraction buffer) usually yields less foam than blending or sonicating. Ideally you work always on ice and as quickly as possible, keep all extracts on ice and get any enzyme assays done as quickly as possible. You can also quickly freeze a 50/50 mix of extract and SDS PAGE sample buffer and keep for SDS-PAGE later. Then you can do the total protein assays (i.e. BioRad) in peace for normalisation later. Enzyme activities can be divided by the protein levels at a later stage to get the specific activity. The frozen sample buffer-extract mixes can be diluted after boiling (which causes a homogenous mixture of denatured polypeptides), prior to loading SDS-PAGE.
Good luck, and best wishes
Jurgen
Frothing can be harmful for certain enzymes, as all the foam bubbles increase the interfase between gas and aquous extract, so a lot of oxygen can enter the liquid and that will help the plant enzyme polyphenol oxidase to cause the typical browning you see when you cut apples or pears and leave the cuts exposed to the air for a few minutes. Some enzymes can be affected during these oxidations and it will reduce activity, although less problems would be expected for western blotting under denaturing conditions. In general, I try to avoid frothing as much as I can. Foaming depends on the extraction buffer (does it have detergents?), the type of tissue (does it have lots of lipids?) and the homogenisation method (do you grind with mortar and pestle, or do you have a blender?). Gentle homogenisation in a mortar on ice (with ice-cold extraction buffer) usually yields less foam than blending or sonicating. Ideally you work always on ice and as quickly as possible, keep all extracts on ice and get any enzyme assays done as quickly as possible. You can also quickly freeze a 50/50 mix of extract and SDS PAGE sample buffer and keep for SDS-PAGE later. Then you can do the total protein assays (i.e. BioRad) in peace for normalisation later. Enzyme activities can be divided by the protein levels at a later stage to get the specific activity. The frozen sample buffer-extract mixes can be diluted after boiling (which causes a homogenous mixture of denatured polypeptides), prior to loading SDS-PAGE.
Good luck, and best wishes
Jurgen
Thank you Prof. Denecke.
We use triton-X as the detergent in the extraction buffer. So is it better to use the detergent after homogenisation? We do have a mechanical homogeniser.
Yes, you can do a 2-stage extraction, first you homogenise without detergent to break open all the cells, then add detergent and homogenise again (very briefly), and indeed you will have less foam.
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