I am isolating proteins and RNA from the same sample (TriPure) and follow the manufacturer's protocol. After the protein pellet is washed 3x in GuHCl/EtOH and 1X with EtOH, I dry it and then dissolve it in 1% SDS at 55C with occasional vortexing for 15min.
Most of the pellets dissolve completely and I noticed the "trick" to achieve that is not to over-dry the pellet. As the protocols states, I remove any insoluble material by centrifugation at 10,000 ´ g for 10 minutes at 2–8 °C.
And here is where the funny part begins - at some samples I got a huge snow-white, paste/jelly-like sediment and almost no free-liquid supernatant. Other samples have no such sediment.
That makes the total protein yield quite different between one sample and another - it looks like the "jelly" traps proteins.
I read that Gu-HCl and SDS can form precipitates (actually there is a publication how to eliminate an excess SDS by Gu-HCl), I was thinking that this white "jelly" could be actually a complex between the SDS and Gu-Cl that remained in the pellet after the final EtOH (Gu-HCL free) wash.
If that is the case, a second EtOH wash before drying of the pellet may remove any remaining Gu-HCL, but I haven't tried that yet.
Had someone experienced the same problem ? How did you solve it ?
I'm not familiar with TRIpure protocol, but don't you think, the white jelly could consist of the genomic DNA?
I though about it Maciej, but because the DNA in theory should have been extracted in the previous step of the treatment of the remaining organic phase I doubt it.
However, if we suppose that previous step didn't work well (e.g. there is DNA in the protein pellet), If I'm not wrong, I believe the SDS can't form a precipitate with DNA by itself (I'm at least sure that 0.5% SDS can be used to dissolve RNA pellets).
However, if there is a salt still present, may be it can precipitate SDS AND the remaining DNA?
I noticed the same problem with my samples and resolve the precipitation problem by centrifuge the samples at room temp instead of 4deg. I was wondering what this pellet was. I've already noticed this kind of precipitate in simple cell lysis with tris-sds buffer at 4 deg, without using the trizol method...
Hello Elise, as what I observed and what I found in various sources that SDS precipitates at low temperatures. I am wondering why the TriPure/ TriZol manuals would recommend performing that step. What they claim is to "remove any unsoluble material", but what may actually happen is that this step precipitates the excess (e.g. unbound to proteins SDS). I am still waiting for their anwer on the topic.
Dear Petar, did you get an answer on the topic? Because I have a problem with the same kind of crystals as you describe, and is therefor very interesting in what you ended up with.
Hello, I have also problems with this protocol. I´m unable to completely disolve the precipitated, even after heating at 50 degrees. When I try to quantify by BCA the yield is very low (around 200ug) .. My starting material is PBMCs (around 5 million per sample).
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