During sonication/lysis after adding the lysis solution, protein extraction results in foam formation. Does this have any negative impact on protein quality? please provide me with suggestions
Thanks,
Hi
Of course, the foam formation during your protein isolation implies that you are denaturing the proteins with the consequent losing of their biological activity.
I recomend you to prevent the foam formation by a gently treatment of your fractions.
Good luck
Hi
Of course, the foam formation during your protein isolation implies that you are denaturing the proteins with the consequent losing of their biological activity.
I recomend you to prevent the foam formation by a gently treatment of your fractions.
Good luck
I agree in part although some of that foaming is due to detergents in lysis buffer designed to lyse cells and if extracting from mammalian tissue internal lipid as well. If at all possible try not to sonicate unless the amount of tissue you extract from is grams in amount in which case it might be unavoidable. Repeated gentle pipetting for smaller tissue quantities might suffice and DNase digestion might substitute for sonication which is principally there to shear genomic DNA
Foaming is BAD really really... denatured protein!! Sonicate at lower power for more time or reposition your probe or change your tube or sample size. Do not allow your sample to foam.
it depends on the tissue and experiments afterwards. If you going to use the Proteins for Western Blotting there's no impact on quality. This is true for Kidney and intestinal membrane fractions. But in all I agree with the last comments.
Hello,
The generation of foam should be avoided at all times as it can not only result in the loss of sometimes large amount of sample, but it can also lead to denaturation of your protein.
Concerning the lysis protocol, I have the following to add:
a) Lysis solution with high detergent content usually involves chemical lysis and does not necessarily include sonication. In fact, sonication should be avoided if it causes the generation of excessive amounts of foam. In the CelLytic B protocol, for example, addition of Benzonase (DNAse) is suggested, and even this is optional and often not necessary.
b) Other protocols for breaking cells such as freeze-thaw, sonication itself and/or usage of lysozyme regularly do not include detergents and thus sonication should not cause excessive amounts of foam to generate. If it does, modify the position of your sonotrode and/or the sonication intensity. The surface of the liquid should only be slightly disturbed with each burst, which will provide movement of the liquid during the process while avoiding the generation of large amounts of foam.
Sonication generally is a reliable way of both cell breakage and DNA shearing at the same time and can help to avoid steps like the addition of DNAse (followed by sometimes lenghty incubation times) or using a disperser tool (potentially generating foam) to dissolve DNA-clots which can form after freeze-thaw. However, heat is being generated during sonication and some proteins can react unfavorably (e.g. becoming insoluble or aggregating) to lengthy sonication procedures ("over-sonication").
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