Hi to all,
I performed a pull down assay of a protein using streptavidin beads attached to a biotinylated oligonucleotide probe, and once the elution step and the boiling step (in loading buffer with SDS and DTT) I found the protein as a dimer in the eluted fraction and as a monomer in the unbound.
How is it possible?
Hi Antonio,
How much DTT did you have in your sample buffer, and was it freshly added? If not, what temperature was your sample buffer stored at, and how long ago was it made?
Hi Antonio, just to add to Mark's answer, are you treating the unbound fraction with SDS and DTT before analysis? Are you doing PAGE or gel filtration?
My guess is that you are seeing a protein monomer in all cases, but it runs differently depending on whether it is fully denatured or not.
hi to all, the concentrations of both DTT and SDS are the same of a classical laemmli buffer.
In my opinion, it is quite unlike that what I see is the protein differently denaturated, because I have also loaded on my gel the protein not treated (so it appears as a monomer). The proteins pulled down and eluted appear as bands havin' a weight consistent with the dimer of the protein.
Another particular is that the protein in the supernatant of each pull down reaction, appears as a monomer.
I have followed the protein by means of western blot, because during the elution step, also the monomer of streptavidin is eluted, and the dimer of my protein has just the weight of the streptavidin monomer
Hi Antonio, this an interesting problem. As far as I know, streptavidin monomers can also bind biotin.
Can you include excess free biotin in your steps to exclude that what you are seeing is the streptavidin monomer that never released the biotin probe or is re-binding your biotinylated probe after the PAGE?
Targeting a bZIP protein by any chance? Coiled-coil domains in these proteins are sometimes resistant to denaturants. Since many sequence specific DNA binding proteins bind DNA as a dimer, and may be monomeric in the absence of DNA, the presence of dimers in your oligo-associated fraction is not surprising. It is somewhat surprising that the dimers are SDS-resistant. How are you performing your elution? Have you tried treating with high salt (>300mM) to disrupt protein-DNA interactions or removing your oligo with Benzonase nuclease? I would expect your protein to resolve back to monomer under these conditions.
Antonio- streptavidin can cross-react with some secondary Abs, especially if present in large quantities. Missed this feature of your previous post- "the monomer of streptavidin is eluted, and the dimer of my protein has just the weight of the streptavidin monomer." Maybe you are only seeing streptavidin monomer in your "bound" fraction. When you perform your blots, make sure you include a lane that has SA beads only (no oligo, no protein of interest), to control for specificity of your Western blotting antibodies.
thank you very much. I have noticed also that the elution of the streptavidin changes if I use or not Biotin or a biotynilated molecule. so is it possible that a biotynilated molecule/biotyn stabilizes the streptavidin from the leakeage?
Yes, Antonio. I have recently observed this elution phenomenon, too. Only some of the streptavidin tetramer subunits will be covalently crosslinked to the support resin. In the absence of biotin, the tetramer is readily disrupted by SDS and boiling. Biotin apparently stabilizes the complex against denaturants, causing far less streptavidin to elute off the beads in sample buffer when biotin is present.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression