I am trying to show an interaction of a protein with RNA in EMSA, on the first day of purification it is showing good interaction in the sense that we are getting clear band, after 2 to 3 days we are not getting clear band, what could be possible reason?
Hi there,
As you mention in your title it seems your protein RNA binding activity is not stable during storage. Then you have to think about improving storage conditions in terms of buffer composition (pH, ionic strength, nature of the buffering agent, presence of additives...), temperature, protein concentration aso... Hope it will help.
In order to preserve the stability of proteins, I usually supplement my purified proteins with 5-10% glycerol. Through experience, you will find out what are the buffer conditions that suit the protein that you are working with.
Hi,
It is not so clear for me whether you want to preserve the protein and used it again for new interaction assay (repeat) or you want to preserve the protein RNA complex.
- If you want to preserve the protein then you need to take the advises above to maintain the best buffer composition which will preserve your protein 3D structure. This would be really depend on the protein. For example I have some experience with protein that very sensitive to oxidation then you might need to dope DTT on the buffer and so on.
- If you want to preserve the complex you might think about crosslink the complex with UV or chemical crosslinking protocol. This will make a covalent bond between your RNA and protein complex which hardly affected by the protein structure anymore. So the complex would be quiet stable in various environment.
Godd luck!
@ Dominique Liger , I am storing the protein seperately, when it is used for emsa after few days it is showing weaker interaction when compared with previous one.
Hi,
do you store your protein at -80° or at +4°C? If you store it at -80°, probably your protein doesn't like to be frozen. In this case, you can try to improve the situation by adding glycerol. With 5-10% glycerol your protein will freeze anyway, but it could be happier, and such percentages shouldn't interfere with your binding essays (I use 5% with my proteins, and they bind DNA). You can increase glycerole up to 50%, your prontein will not freeze but it could be a problem for your interaction.
Did you check if your protein precipitate after storage? If it's like that, It means that it's unhappy in that buffer. You can try to adjust pH or salt concetration, before changing everything. If your buffer has reducing agents, like DTT or beta-mercaptoethanol, I suggest to add fresh ones just before the assay: their effect lasts only few hours and it could be crucial for your protein to interact.
I also suggest to check if your protein aggregates after storage. You can easlily verify it by size exclusion chromatography. Aggregates could also prevent the interaction with the substrate.
Hope it could help!
hi patrizia,
i am storing in -20 and it is not precipitating. I added glycerol too. I am getting interaction but no as good as previous one I mean after storage.
Did you have glycerol in the buffer when you got good results? Same % that you used for storage?
I also guess you have no reducing agents in your buffer. If the protein doesn't precipitate, and the interaction is not influenced by glycerol or reducing agents, I would say that the protein is damaged by freezing. Sometimes It can happen. A colleague of mine is in the same situation, she couldn't overcome the problem so she purifies fresh protein every time she has to do an experiment.
Luckily each protein is different from the others, probably you can find a solution just playing a little bit around the buffer conditions.
Yeahh, it might be the possible reason. Thank you all for your suggestions.
Hi,
You had better avoid thaw-freeze cycles, so before their storage at -80, you should make small aliquots