Dear Laura,

you can use ultrafiltration with 3 kDa colums to get rid of the free QDs or PD10 colums.  When the dye or the protein binds unspecifically to the membrane of the centrifugation units try to block the membrane first e.g. with BSA before ultrafiltration or try a different membrane e.g. Hydrosart instead of PES or CTA.

Best,

Gaby

There are potentially 3 categories of products: (1) peptide-coupled quantum dots, (2) uncoupled quantum dots, and (3) uncoupled peptide. It should be easy to separate (1) and (2) away from (3) because of the large difference in size, using gel filtration, centrifugation, sucrose gradient ultracentrifugation, dialysis or ultrafiltration (if the non-specific sticking problem can be solved).

It would be more difficult to separate (1) and (2) because they are so similar in properties. The need to do this could be avoided by making sure all the quantum dots have peptide attached by using a large molar excess of peptide over quantum dots. However, a possible approach to the separation would be to make the peptide with an attached affinity tag, such as hexa-histidine or biotin. Then, an affinity resin such as immobilized Ni2+ or streptavidin, respectively, would be used to pull down (1), leaving (2) behind, Finally, (1) would be eluted from the resin by imidazole or heating+biotin, respectively.

Gabriele and Adam, thanks a lot for your suggestions.

From most of the papers, I understand that they are not doing any further separation between peptide-coupled QDs and uncoupled QDs, and therefore assuming a high efficiency for the EDC coupling. Since we are still in the optimization steps, I am no sure if we can make this assumption. but we can try to increase the peptide-QD ratio.

Best regards,

Laura