I have protein extraction from mamalian tissue,I want to do sonication to get rid of DNA and RNA then I will measure protein concentration by using nanodrop,and finally I will run on SDS-PAGE.
I'm looking for the right tempreture,cycle,and protocol for sonication without affect on the protein.
Most sonications are performed on ice. Some proteins can handle more sonication than others. The only way to determine the best conditions is to try several sonication times and run a Western blot for your protein of interest. For some proteins, we observe a sonication time-dependent loss of protein in our gels. Sonication also helps to remove DNA-bound proteins as well as opening up highly structured proteins and complexes which may assist in antibody-epitope recognition in your Westerns.
I'm confused. Why exactly do you want to remove DNA and RNA in this manner?
In all honesty, RNA will more or less remove itself as soon as you homogenise the tissue, just via endogenous RNAses, and DNA degradation (while not nearly so fast) will also occur via liberated nucleases. Moreover, neither oligonucleotide will really affect downstream SDS-based denaturing protein gels anyway.
So I'd just dissolve the extract in SDS PAGE loading buffer (and maybe boil for a couple of mins) then run it.
Dear Eric,
As you said first you measure protein concentration. Than you boil the sample with classical Laemli buffer. The glue means you have to much DNA. Than you sonicate it until the foam with small bubbles will apear and the sample will become hot. The sonication could be replaced by insulin-needle passing through until the glue will dissapear.
keep fingers cross - jan
Hi Eric, for removal of nucleic acid contaminant by sonication, two sonication treatment may be needed. sonication treatment for 10 cycles of 30 s pulse with 30 s interval on ice will be good and does not affect on your protein.
regards
Hi Eric,
For sonications, one trick could be that to increase the number of duty cycles (rather than the sonication time).
Also, the blast usage is also crucial. Albeit it is not very well standardized amongst sonicator brands, typically an arbitrary level of 2 to 3 would be sufficient.
By the way, Would you quantify your proteins using Bradford assay or others (on top of nanodrop)?
Most sonications are performed on ice. Some proteins can handle more sonication than others. The only way to determine the best conditions is to try several sonication times and run a Western blot for your protein of interest. For some proteins, we observe a sonication time-dependent loss of protein in our gels. Sonication also helps to remove DNA-bound proteins as well as opening up highly structured proteins and complexes which may assist in antibody-epitope recognition in your Westerns.
Hi Eric
I think you can sonicate your cell suspension for 30s pulses on ice (this ice bit is important) for 5-6 times or until you see your cell suspension is no more cloudy (Please use protease inhibitors if you're using a buffer). Although I've done this with mammalian cells and it works absolutely fine, but yes as Dr. John has mentioned, it might affect your final protein output as different proteins handle sonication differently. After your sonication, do a hard spin for 10 mins, you can get your protein in the supernatant which you can boil with some Laemli buffer for your SDS-PAGE run.
For a good proteins separation in addition to buffer RIPA (with proteases inhibitors, phosphatases inhibitors and EDTA) the samples were sonicated on ice to a 100% amplitude, pulse 50%, for 15 seconds. Then, mix de samples in shaken in cold conditions and finally spin during 15 minutes at 4 degrees. Supernatant fraction is ready to proteins quantification.
I hope the below papers to help you.
10.1016/j.ifset.2010.07.002
10.1016/j.jcs.2009.06.006
For sonications, one trick could be that to increase the number of duty cycles (rather than the sonication time).
Also, the blast usage is also crucial. Albeit it is not very well standardized amongst sonicator brands, typically an arbitrary level of 2 to 3 would be sufficient.
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You have to consider the output power of your sonicator.
Usually, you'll simply need some preliminary experimentation.
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