Hi everyone,
I'm trying on-bead RNA pulldown of a single, fluorescently labeled protein from a cell extract. I use Qiagen Ni-NTA agarose beads which I coat with His-streptavidin first and then with biotin-labeled RNA. The thing is, majority of my protein of interest leaks into the beads super quickly, lowering the assay-available amount to interact with the RNA. It does not just get inside the bead and distribute evenly throughout it, instead it forms a clean ring, right under the bead "surface". Happens in the RNA(-) control, too. Also, when I incubate my cell extract with the RNA first and add it to strep-coated beads as a last step, I see the interaction. However, I really want to avoid doing the assay this mixed up way for high-throughput reasons. I suspected pH issues but I use regular, published recipes, which I re-made to double check and it did not help.
Could someone explain what kind of sorcery is this and how to prevent it from happening?
Thank you!
Natalia
If this is non specific binding to the bead matrix then you could try blocking the non specific binding sites with some other protein like BSA before doing the specific adsorption
Hi Natalia
If you know the sequence of your protein, please check the isoelectric point of the polypeptide. It may be due to instability because your protein could be close to the pI. If this is the case, you can easily change the pH of your binding buffer to ensure 1.5 units far away from the pI. If the protein is far away from the pI, it will be charged and remain soluble during your assay.
On the other hand, RNA-binding proteins love high salt concentrations. Some of them are only stable at 500 mM NaCl or higher. Please check the ionic strength of the buffer.
Hope it helps. Best
Paco
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