Selecting a secondary antibody for QDs

Hi everyone,

In our phone meeting last week we briefly touched the issue of picking a secondary antibody to conjugate with QDs. Of course, the discussion actually started when we were in Georgia. Here is the original suggestion from Beth’s student Julia

<“Goat anti-mouse IgG2b” but we can change the goat part if necessary.>

I realized that my lab will be frequently using mouse hippocampal neurons from now on (although we will continue to use anti-mouse secondary for Drosophila). So for secondary antibody my lab may also need to choose “Goat anti-rabbit IgG”. But I understand that Beth’s lab will also be using single muscle cells from rabbit (if I recall correctly). In view of this, we might need to have two secondary antibody options instead of one. I understand that this would mean more work and expense for Jessica’s lab. Your thoughts will be much appreciated. I hope that we can arrive at a decision soon.

Best regards,

-Ge [via email]

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5 Responses to Selecting a secondary antibody for QDs

  1. Jessica says:

    I apologize for missing the meeting. I am happy someone from our group attended though.

    Regarding antibodies, it is best if Beth and Ge agree on one and we can move forward with conjugating our particles directly to that secondary. If needed, we can do one for each of you, but each chemistry would have to be optimized and this would take longer.

    Also, can you tell me how the DSPE-PEG dots worked for microinjection vs. the larger PS-PEG dots we sent?



  2. Beth says:

    On the issue of secondary antibodies for targeting qdots. We are currently quite busy with the microinjection work and a bunch of rabbit psoas immuno work that we are doing with the Alexa 488 dye. This latter work is interesting because we find that we can do immuno staining on unfixed “glycerinated” muscle fibers, so we can subsequently cause them to contract by adding calcium and ATP to the solution, and then image them in the contracted state. Even just with Alexa and confocal, these results are looking interesting, and may be even more interesting if we can get STORM images. We expect to send some slides to GA early next week.

    So — this is all to say that we are not in a huge rush to receive the targeted qdots for our rabbit muscle cells. We have plenty of work to keep us busy. On the other hand, the glycerin-permeated rabbit muscle fibers could be a good test-bed for the targeted qdots because the dots can get into the permeabilized cell membrane, go to the target, and unbound dots can be washed away. Then we should still be able to cause contraction and potentially see a biological process (maybe in action, maybe just start and end points).

    If Ge’s group is ready to work with Goat anti-rabbit IgG qdots on a shorter time scale, then that should be the higher priority. But at some point we will want Goat anti-mouse IgG2b for the rabbit muscle.

    — Beth

  3. Ge says:

    Hi all,

    Sorry for the silence. Just came back from a seminar travel to Buffalo, NY.

    We are moving along well with the hippocampal neuron culture. So if Goat
    anti-rabbit IgG qdots is ready, we can test it soon (I recall that Shumin
    Nie’s groups published their QD immunostaining protocol in an article in
    Nature Protocols). I will get back to you with more details. I have teaching
    at 1:30PM.



  4. Beth says:

    This would be fine with me. Goat anti-rabbit IgG first.

    — Beth

  5. Jessica says:

    OK. Thanks, we are meeting this Friday and will talk about this. Also,
    do you have a specific supplier cat # you want us to use? My standard
    would be JacksonImmuno….


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