I am digesting ribosome with RNase A to remove rRNA and release the ribosomal proteins. When I run a 1% agarose gel after reaction, compared to my control (without digestion), in the experiment lane a smear of RNA is showing at the extreme top of the gel. What might be the reason? There are degraded RNA products at the bottom of the gel, but what are those at the top?
General comment:
You are missing an "RNase A only" control. That would be the simplest source of the additional band. Eliminate this option before anything else.
Global suggestion:
I assume the samples are NOT phenol/chloroform extracted and are run on a NATIVE TAE or TBE agarose gel. Correct? So the first thing to do is either to ph/ch extract the samples (the proper way) OR add a denaturing reagent to the loading buffer so you can judge the size of your RNA more reliably (for a quick and dirty visualisation). For similar setups I routinely add SDS to 0.1-0.5% before loading on a native agarose gel (yes, it works). This comes with several warnings: (1) Don't forget to add SDS to the size markers if you use any; (2) SDS itself binds EtBr or SYBR green, visible as a VERY bright fast-migrating band so include an SDS-only sample alongside; (3) make sure you run the sample far enough as the SDS front "chelates" the EtBr; (4) load the samples slowly as the SDS makes them more prone to leaking and bubble formation.
Alternative considerations once top comment is dealt with:
Even the magical RNase A will only digest accessible and single stranded RNA. In native ribosomes much of the rRNA is folded and hidden. Therefore, it is possible that after digesting the accessible RNA, the structure of the ribosome breaks down and the proteins aggregate non-specifically. The "heavy" staining is then generated by the protected rRNA fragments in the aggregate.
You can try any combination of the following:
* reducing amount of ribosomes in the digest.
* increasing the temperature of the digest. RNase A can cope with 60oC. Obviously, the ribosomal proteins may not fare as well, but I don't know what you are planning to do with them.
In addition, we repeatedly found that high doses of RNase A cause many things to become non-specifically sticky. We mostly observed it in co-immunoprecipitation of RNPs and never bothered checking out why or how. It may be worth simply titrating down the RNase A concentration, perhaps with a longer incubation and/or higher temperature.
Hope this helps, and please post the answer once you figure it out.
Hi Parijat
Is it possible for you to upload the gelpic...it will be more clear to the RG users
bye
Lane 1 is without RNase A, Lane 2, 3,4 are with RNase A, lane 5 6 are without RNase A.
I think that it isn't RNA. RNA it isn't so heavy. This is some residue of your protocol.
It is ethidium bromide staining , and ribosome consists of rRNA and proteins only.
It is undigested RNA attached to high molecular weight proteins.
General comment:
You are missing an "RNase A only" control. That would be the simplest source of the additional band. Eliminate this option before anything else.
Global suggestion:
I assume the samples are NOT phenol/chloroform extracted and are run on a NATIVE TAE or TBE agarose gel. Correct? So the first thing to do is either to ph/ch extract the samples (the proper way) OR add a denaturing reagent to the loading buffer so you can judge the size of your RNA more reliably (for a quick and dirty visualisation). For similar setups I routinely add SDS to 0.1-0.5% before loading on a native agarose gel (yes, it works). This comes with several warnings: (1) Don't forget to add SDS to the size markers if you use any; (2) SDS itself binds EtBr or SYBR green, visible as a VERY bright fast-migrating band so include an SDS-only sample alongside; (3) make sure you run the sample far enough as the SDS front "chelates" the EtBr; (4) load the samples slowly as the SDS makes them more prone to leaking and bubble formation.
Alternative considerations once top comment is dealt with:
Even the magical RNase A will only digest accessible and single stranded RNA. In native ribosomes much of the rRNA is folded and hidden. Therefore, it is possible that after digesting the accessible RNA, the structure of the ribosome breaks down and the proteins aggregate non-specifically. The "heavy" staining is then generated by the protected rRNA fragments in the aggregate.
You can try any combination of the following:
* reducing amount of ribosomes in the digest.
* increasing the temperature of the digest. RNase A can cope with 60oC. Obviously, the ribosomal proteins may not fare as well, but I don't know what you are planning to do with them.
In addition, we repeatedly found that high doses of RNase A cause many things to become non-specifically sticky. We mostly observed it in co-immunoprecipitation of RNPs and never bothered checking out why or how. It may be worth simply titrating down the RNase A concentration, perhaps with a longer incubation and/or higher temperature.
Hope this helps, and please post the answer once you figure it out.
Dear Anna,
Very nice response! I agree with your assessment.
Dear Parijat,
I've observed large aggregates when I add RNase A to ribosome samples. Rnase A can't digest the rRNA completely. On top of what Anna mentioend...if you add EDTA to chelate any magnesium that might be around this will also help to complete the digestion. Best of luck.
I completely agree to Anna's analysis. I guess it 's very possible that the stained band-like stuffs contain fragmented RNAs that are in complex with proteins.
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