In a previous post, I explained problems encountered with a quantum dot (Qdot)-conjugated secondary antibody. Since then, I have received a replacement Qdot-conjugated secondary antibody (Q22085: Donkey anti-Mouse IgG (H+L) Secondary Antibody, Qdot 625 conjugate) and have had success with labelling tubulin of HeLa cells with Qdots, Hurrah! This is clear from microscope images, where tubulin has been dual-labelled with both an organic dye (Alexa Fluor 488) and the Qdot-conjugated secondary antibody (Figure 1).
As mentioned before, one of the things that has been bugging me about the SmartFlares is that they’re not where anyone expects them. EMD expect them in the cytosol (we don’t see them there), I expected them in endosomes and they weren’t really there either. So what could this mystery compartment be?
We’ve so far, not seen any response of the VEGF SmartFlares to treatment of our cells with DMOG. Combined with the punctate distribution of the signal, we have no evidence that the SmartFlares make it to the cytosol in 18 hours. As we’re not sure if the problem lies in the SmartFlares or the delivery, I microinjected the SmartFlares directly into the cells thus bypassing the need for endosomal uptake.
I recently discussed the SmartFlare project at a joint Sée and Lévy Group Meeting, partially extolling the virtues of Open Science but mostly focussing on the data acquired so far and some of the proposed experiments.
Regarding the SmartFlare project, the three big questions that formed the basis of the talk were:
- Why do only some cells take up SmartFlares?
- Where are the SmartFlares?
- Why are the VEGF and Scrambled SmartFlares fluorescent in puncta?
Below are some of the interesting points that were made during the meeting. Replies at the time (as best as I can remember them) in green:
In order to investigate why the SmartFlares seem to be fluorescent in puncta within the cells, we designed an experiment to try to inhibit endosomal maturation, thus (hopefully) preventing SmartFlare processing and release of fluorescence.
With the Undergraduate project reports now marked, Gemma has kindly agreed to post her project report on the blog. You can find a pdf of the report here.
As most of the methods and results are already on the blog, a slightly abridged and edited version of the discussion section is replicated below: