I have lysed my cells using 8 M urea, but I noticed that the pellet is very viscous and it was hard to get rid of pellet and take supernatent. Why did this happened? and is it affected on measure protein concentration?
The viscosity is probably due to released DNA. You can sonicate the sample (maintaining the sample ice-cold), or shear it by running it up and down to break up the DNA.
Lysis using 6-8M urea is a standard method. If you are not getting proper pellet then you need to do certain changes in your protocol. First change you can do is to increase the rpm during centrifugation followed by increase in the time. This allows the matters with different density to settle down and make a firm pellet. All the best. :)
I do not know if you add detergents, but they are necesseray. To inscrease solubility of your protein, you need to perform one or two cycles of freeze/thaw. You could add some buffer in order to increase the dilution
The viscosity is probably due to released DNA. You can sonicate the sample (maintaining the sample ice-cold), or shear it by running it up and down to break up the DNA.
i mainly assume that you lyse the sample for proteome analysis. The viscosity problem is mainly because of DNA and sonciation would help to get rid of the viscosity problem. Also even if you use detergent you will still see this problem. however as mentioned above it is important not to heat up the Urea sample since this can modify the peptides in case you are looking to analyze them by MS. another alternative for Urea is guanidium chloride which is also good in case you want to perform insol digestion at 37 C whereas with Urea preferably digestion is performed at room temperature. Further it would be beneficial not to centrifuge down the pellet and still keep them in the solution for digestion so that the loss of membrane proteins in regular in-solution protein digestion protocols is kept to minimum.
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