Hi to all,
I'm trying to characterize the alleged interaction between a protein and liposomes having a specific ratio of anionic ang zwitterionic phospholipids.
I have performed both a control run with liposomes and protein alone and i follow the signal at 280 nm. Both the liposomes and the protein have an emission signal, and these signal are quite distinguishable. Furthermore the signal of liposome has a very high intensity compared with the one of the protein.
When I perform the run on the mixture, in which both the protein and liposomes have the same concentration as in the control samples, I see that the protein signal decreases in intensity compared with the one in the control sample, the peak of liposomes has the same intensity as in the control, and I don't see any variation in the peaks: nor a delay or an anticipation of the peaks of the protein and liposomes.
Do you have any experience with these kind of experiments?
Thank you
The 280 nm absorbance of the liposomes probably results not from a chromophore but from Rayleigh light scattering (turbidity). You can check this by taking an absorbance spectrum of the liposomes. If it is due to light scattering predominantly, you will see the absorbance increase steeply as the wavelength decreases. In contrast, the protein absorbance is probably due predominantly to aromatic amino acid side chains, resulting in a peak at about 280 nm (unless it is highly aggregated, in which case the peak could be shifted to shorter wavelengths due to light scattering).
If your protein sticks tightly enough to the liposomes to elute with them, you may not be able to distinguish the weak protein absorbance on top of the strong light scattering-derived absorbance of the liposomes, but you will see a decrease in the absorbance of the protein peak due to the reduced amount of free protein.
To make it easier to detect the liposome-bound protein, you could attach a label of some sort to the protein, such as a fluorescent dye or radioactive tag, or you could run the liposome fraction on a SDS-PAGE gel and stain for protein (the easiest method), or you could use immunological detection (Western blot or ELISA).
dear Antonio,
I do not understand how do you managed to see any emission peaks at 280 nm in protein-liposomal mixture? What is emissing? Perhaps, you mean light absorbtion, in this case see Dr Shapiro's comment.
If I were you, I would separate your mixture by gel-filtration (as you indicate in the question), for instance in Sephacryl column, and then I would measure light absorbtion of every fraction - probably, it will be protein-bound liposomes, liposomes and unbound protein. You can hardly separate protein signal in its mixture with highly scattering microparticles (liposomes).
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