Native Gel for QDs and Peptides - (Aug/28/2006 )
Hi
I'm trying to use gel electrophoresis to separate quantum dots and quantum dots-bound peptides. The particles can be either anionic or cationic and the peptides are ~20aa long. There can be many peptides bound to one nanoparticle.
Based on the purpose of each reagent in a typical SDS-Page gel, I was thinking it is best I use a native page gel so the SDS does not interact / precipitate the cationic particles. Also I decided to remove SDS and mercaptoethanol/DTT from the sample buffer solution, since the peptides do not really need any further degradation.
Is my decision reasonable? I'm pretty new to this so I'm not too sure. Any other recommendations?
Thanks.
You may want to use low % gel, like 6 or 8 % and alterantive running bufffer for cationic QD (MOPS-HOAC, pH 5). In that case, you nned to reverse the electric field direction so that the band will move toward bottom. For your purpose, agarose gel may also work.
since you have a mixture of anionic and cationic peptides, using a native gel will be problematic. you will either have to run 2 gels (one basic pH separation and one acidic pH separation, with reversed electrodes) or you will have to ensure that your running gel pH is higher than the highest pI of your peptides (or lower than the lowest pI, with electrodes reversed).
another problem: are all your peptides the same size? if so you may have trouble separating them.
therefore, i would recommend that you run your peptides on isoelectric focusing (ief) gels. they will be separated based on their pIs and you should be able to separate all of them, regardless of size.
unfortunately, this generally requires apparatus designed for this purpose. there are ways around this requirement but it is best to use equipment designed for the purpose.
I think I might just run both separately, as suggested by genehunter-1. However, I'm not so sure about running the cationic one. So I have to change the running buffer pH to about 5 (MOPS, HOAC). How about the pH of the gel? Will I need to change that too? I am probably going to use a continuous gel.
If the purpose is to demonstrate surface charge properties of peptide coated QDs, I would cast an horizontal agarose gel (1% in TAE or TBE) with combs placed in the center. Load your samples, cationic peptide, cationic peptide-QD, anionic peptide, anionic peptide-QD, as well as QD. Run the gel and follow the QD fluorescence. You will see the two QDs go different directions. Its simple and effective.
omg! that sounds like a great idea! thanks for that. i will go try it out in these couple of days!
some guys in my lab use this technique, and it works quite fine. PAGE can be a problem anyway, because QDs are rather large. so 0.7-1.2% agarose gel should be used. only problem that can remain, are uncharged QD. they do not move at all, and are washed out of the pockets after the run
only with very careful handling after the run (no need to stain, if fluorescent properties remain), they'll stay in their place. what pH should I be using in this case though? I tried it yesterday with normal TBE buffer (ph ~8.4) and the anionic one moved well. However, the cationic ones were stuck in the well. Some cationic ones were attracted to the cathode side but some were attracted to both cathode and anode sides (maybe those are actually neutral).
i guess my question now is... what is the pH I should use? I'm thinking of using ~pH 7 but does TBE work well as a buffer at 7? If not, are there any other buffers I can use? I'm thinking of using a sodium borate buffer (boric acid/NaOH) at pH 7 for making gel and running solution.
I would prefer TA without EDTA over TB, as boric acid and EDTA may interact with polycation peptide chains on QDs. The pH is not a very big issue for cationic QDs, as Lys and Arg have pKa 9.8-12 on their side chain groups and will remain postive with this buffer. They will move to the opposite direct as anionic QDs does.