I am working with a florescent compound that is biotinylated. When strepdavidin is present in the system, the binding of the protein with the compound quenches the florescence. I need to perform an experiment in presence if streptavidin, however I need to measure the florescence of the compound. The problem I am facing is because of the florescence quenching when streptavidin is present in the same system. How can I effectively disrupt the biotin-streptavidin interaction so that I would be able to quantify the compound by measuring the florescence. I found few methods like heating and pH alterations, however mild conditions are reversible and harsh condition might affect my compound too. Is there any way to effectively break the biotin-streptavidin interaction and separate them? Both are in soluble state in the system. Any help would be highly appreciated. Thank you.
Dear Ankit Pandeya,
1.A short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used and your compound of interest might be not harmed.
2.We use methanol to elute the biotin/Streptavidin compounds but this might affect your experiment too much.
3.You can also try to add an excess of biotin so that the biotin competes with the biotin bound to streptavidin.
4.If all this does not work, it might worth to try the TAPS label from BioTeZ. The TAPAS label is a chemical tag which is recognized by an antibody and can be used as alternative to Biotin/Streptavidin.
Best, Janko
Hi Ankit,
Sorry to say but there is no mild condition exist to disrupt the biotin-streptavidin bond. The only way working in my practice is adding concentrated HCl. I suggest you to figure out a competition style measurement, if possible.
Best,
Karoly
I would look to use an alternative like Strep Tactin or monomeric avidin.
Alternatively, you would need to add an appropriate volume of 100 mM Glycine-HCl pH2.5, then immediately desalt using a sephadex g-25 spin column. The vast majority of the strepavidin will be in the flow through. Quickly transfer to a fresh eppendorf tube and add 0.5M HEPES pH 7.5 to neutralise the HCl, then spin long and hard to elute your compound.
Thank you so much Dr. Brand, Dr. Liliom and Dr. Stanley for the valuable suggestions. I think for my kind of experiment adding high concentration of free biotin and heating might work, doing so heating might break the interaction and free biotin might compete out my compound during cooling. I will try this and see. Thanks all for the suggestions.
May be this study would be of help:
doi.org/10.1016/j.bbrc.2017.09.168
Excess of biotin alone definitely does not work. The best results were obtained using 0.4% SDS, 95C 5 min heating and 0.25 mM Biotin.
Direct answer to your question - short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can, therefore, be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-uses of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid-phase applications in fields, such as microfluidics and nanotechnology.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins