2
$\begingroup$

I am using Neutravidin Agarose beads to isolate biotinylated RNA. The thing is, in the manual it says to use 8M Guanidine-HCl, pH 1.5 for elution biotinylated molecules. As far as I know it is a very harsh elution buffer. Do you think it would effect my RNA? I have isolated my RNA with TRIzol which also contains Guanidine salt but the concentration is not that high.

As a second question, do you know other ways to elute my biotinylated RNA from beads?

Thanks!

$\endgroup$
4
$\begingroup$

The biotin-streptavidin interaction is extremely strong, one of the strongest (if not the strongest) non-covalent interaction between biomolecules. So you'll need pretty harsh conditions in any case.

Extreme pH values are generally a very bad idea for RNA. I'm pretty sure that the conditions you mentioned are not meant for elution of nucleic acids. Such low pH values (and high pH values as well) lead to a quick hydrolysis of the phosphoester bond of RNA, you can read more about the mechanism of that in this review.

A quick Google search lead me to the following protocol:

To dissociate biotinylated nucleic acids from Streptavidin-Coupled Dynabeads®, incubate the beads in 95% formamide + 10mM EDTA, pH 8.2 for 5 minutes at 65°C or for 2 minutes at 90°C.

This sounds reasonable to me, as formamide is also commonly used in sample buffer for denaturing gel electrophoresis of RNA. I haven't done anything like that myself, so I can't guarantee that this would work in your case.

$\endgroup$
  • 1
    $\begingroup$ Hey, thanks a lot for your answer. I found an other solution. Instead of destroy the bond between biotin and Neutravidin which is obviously so strong, I am going to cleave the bond between biotin and RNA which seems more logical for downstream experiments. The biotin that I am using is binding to RNA with disulfide bonds. So, I am going to use 100 mM 2-mercaptoethanol to achieve it. What do you think about it? $\endgroup$ – golgicik Feb 6 '14 at 14:06
  • 1
    $\begingroup$ @golgicik Cleaving at a different point is the easiest way if you don't need the biotin later and only use it for purification. I can't comment on the practical aspect as I haven't done something like that myself. $\endgroup$ – Mad Scientist Feb 6 '14 at 14:07
  • $\begingroup$ I will use RNA in RT-qPCR so I do not need biotin. Thanks a lot for your input! $\endgroup$ – golgicik Feb 6 '14 at 14:08
  • $\begingroup$ i guess pH 8 won't be good for RNA. Mercaptoethanol or DTT would be a decent option.. I am not sure about this but you may use proteinase-K as well.. It would destroy streptavidin and other protein contaminants. $\endgroup$ – WYSIWYG Feb 7 '14 at 7:16
  • $\begingroup$ I'm way late to comment, but thought I should add that I was able to displace biotinylated protein from avidin with a large excess of free biotin. I can't remember the exact concentration, but it was pretty high, and I had to let it sit overnight. $\endgroup$ – user137 Oct 16 '14 at 22:30

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.