Hi to all,
I'm trying to study a protein DNA complex.
when I try EMSA i have positive results with the nuclear extracts, while with the recombinant protein I have never seen any interaction.
On the contrary when pull down assays with streptavidin magnetic beads are performed, I have good result both with recombinant and endogenous protein.
I know for sure that the protein that binds the dna probe within the nuclear extracts is not post translationally modified, because I performed a previous trypsin Digestion and a Database search for PTMs.
So i'm wandering if the positive results with the recombinant protein and pull down are due to a sort of crowding effect given by the beads, that simulate the environment of the protein in nuclear extracts.
Thanks
The question is, does crowding by other proteins increase the binding of a protein to its DNA binding partner? I can't claim any experience with this topic, but my opinion is that the answer is no, in general.
My reasoning is that the presence of other proteins does not increase the effective concentration of the protein of interest, which is the parameter that determines how much of the complex forms. If anything, crowding, if it were severe enough, might even have the opposite effect, reducing the apparent affinity by restricting the free diffusion of the protein, making it less likely to encounter its target.
There may, however, be conditions under which the presence of other proteins might have the enhancing effect. For example, some proteins are unstable when very dilute. Other proteins (people often use bovine serum albumin) can have a stabilizing influence. As another possibility, some of the other proteins in a nuclear extract may influence the structure of the DNA, for example by changing its degree of supercoiling, that may increase the affinity of the binding interaction. Or some of the other proteins may form a complex with the protein of interest, and this complex may have a higher DNA affinity than the protein of interest has by itself.
Hi there,
Indeed magnetic beads are increasing local concentration of the ligand and therefore favor the formation of the complex with protein. This effect doesn't exist in EMSA experiment which may explain weaker or no interaction observed. The fact that you get something with nuclear extract and nothing with purified protein tends to prove there may be factors in the extract stabilizing the complex.
ok, thank you for the answer. But among those factors stabilizing the complex you mentioned, there could be the crowding of other proteins within the nuclear extract that are missing with the recombinant protein alone?
The question is, does crowding by other proteins increase the binding of a protein to its DNA binding partner? I can't claim any experience with this topic, but my opinion is that the answer is no, in general.
My reasoning is that the presence of other proteins does not increase the effective concentration of the protein of interest, which is the parameter that determines how much of the complex forms. If anything, crowding, if it were severe enough, might even have the opposite effect, reducing the apparent affinity by restricting the free diffusion of the protein, making it less likely to encounter its target.
There may, however, be conditions under which the presence of other proteins might have the enhancing effect. For example, some proteins are unstable when very dilute. Other proteins (people often use bovine serum albumin) can have a stabilizing influence. As another possibility, some of the other proteins in a nuclear extract may influence the structure of the DNA, for example by changing its degree of supercoiling, that may increase the affinity of the binding interaction. Or some of the other proteins may form a complex with the protein of interest, and this complex may have a higher DNA affinity than the protein of interest has by itself.
You could easily quantify the binding affinities with recombinant protein and nuclear extracts by using MST (MicroScale Thermophoresis), in free solution, without any surface immobilization. In the review attached, the binding affinities for a protein-protein interaction with purified proteins and in cell lysate were basically identical, however, we also see differences if other components present in the cell lysate interact with one of the binding partners. Comparing binding affinities in buffer and in cell lysate thus provides interesting insights into biomolecular interactions.
You asked, "...
the positive results with the recombinant protein and pull down are due to a sort of crowding effect given by the beads, that simulate the environment of the protein in nuclear extracts.
Is it correct to consider a protein nuclear extract more crowded than a solution of a recombinant protein? - ResearchGate. Available from: https://www.researchgate.net/post/Is_it_correct_to_consider_a_protein_nuclear_extract_more_crowded_than_a_solution_of_a_recombinant_protein [accessed Jul 20, 2015]." If you are interested in cell physiology (we all should be) then I suggest that you calculate the inter-molecular distance between the macromolecules in your prep and the corresponding distance between macromolecules in cells, or more precisely, the cell organelle in which y=the protein of interest resides.
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