Hi Everyone
I'm working on Targeted drug delivery using Aptamers. I synthesized PLA-PEG-COOH and afterwards I tryed to link Aptamer via linkage of -NH2 in Aptamer and -COOH in PEG. first I dissolved the polymer in dionized water and after 2 Hours activation with EDC and NHS I added Aptamer and stirred it for 5 hours.
In order to get the confirmation of PLA-PEG and aptamer conjugation I run it on agarose gel and evaluated with gel doc but I couldn't detect any sign of aptamer on gel (Im sure that i had not lost aptamers during the conjugation process).
why has this happened and what is its reason?
Could you please offer some other ways to evaluate the confirmation of aptamer linkage except agarose gel?
thank you
Is it possible that all the aptamer molecules have attached to polymer? I mean there was no free (non-conjugated) aptamer or concentration of free aptamer was lower than gel-doc method detection limit. My advice is to use aptamer with fluorescent label and to test different polymer-to-aptamer ratios. Using spin collumns or centrifuge filtration units with MWCO lower than Mw of your polymer you can isolate non-bound aptamer and quantify it (another methods to isolate non-bound aptamer are gel filtration, dialysis, ultracentrifugation, precipitation of polymer by suitable chemical...) Also, you can extensively wash polymer and measure its fluorescence to confirm attachment of aptamer. Aptamer without terminal NH2 should be used as control because non-specific attachment of aptamer to polymer is possible.
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