• Created by Unknown User (teague@mit.edu), last modified by Unknown User (lylaatta@mit.edu) on Jul 09, 2014 16:26

Protein receptor group

BCR group

 

From Kathryn:

Anti-IgM:
We were planning to use cytometry and/or microscopy to verify that the BCR localizes to the membrane. Based on that application, the anti-IgM should bind to a region of the IgM outside of the membrane. Since we weren't sure whether any of the monoclonal antibodies would bind that region, we chose a polyclonal antibody. We chose a biotinylated version because we wanted the flexibility to do Western blots using the anti-IgM. In case we didn't see membrane localization, a Western blot would allow us to confirm that IgM is actually being produced inside the cell. I'm not sure we need the biotinylation though if you're planning on ordering different secondary antibodies. We chose rabbit in particular because it had the shortest arrival time - it seems that polyclonal antibodies can take up to a week to arrive.

Anti-Syk:
We were planning to use anti-Syk exclusively for Western blots. We want to determine whether or not our HEK293 cells express Syk (and at what level) in order to determine whether it will interfere with the BCR. (In case you're wondering, we weren't planning to run the same test with Lyn for a couple of reasons. There are multiple proteins that perform the same function as Lyn so blotting for Lyn wouldn't necessarily tell us if we can expect cross-talk problems. Also, using endogenous Lyn as part of the BCR rather than introduced Lyn shouldn't make a difference for Syk recruitment.) The reason we chose this particular anti-Syk was because it wasn't specifically against phosphorylated Syk. We weren't sure if anti-phospho Syk would necessarily give us an accurate measure of how much Syk was being produced by the HEK293 cells, since not all of the Syk in the cells might be in phosphorylated form. We chose biotinylated because we figured that since the anti-IgM we wanted was biotinylated we wouldn't have to reorder streptavidin Western blot supplies. That said, we don't necessarily need biotinylated if you're planning on ordering different secondary antibodies.

 

From Lyla:

So the antibodies that I have sent you are anti-LirlB2 and anti-PirB antibodies that we want to use in our first experiment testing the membrane localization of the receptors. We are going to express the receptors and have the antibodies bind to them and then look for membrane localization of the antibodies using microscopy. Initially, we were going to use biotinylated antibodies so that we could use the streptavidin-fluorophore conjugate to image them. However, we couldn't find biotinylated antibodies from abcam so we decided that we would just use antibodies conjugated to fluorophores - these are the ones I linked on the orders page on the wiki. 
I was talking to Kathryn earlier about how they're going to compare the amounts of endogenous and exogenous Syk in HEK293 and I think that it may be a good idea for us to run a similar experiment for cofilin i.e. run a western blot of endogenous cofilin and TRE:cofilin-eYFP under different levels of dox. I looked into primary anti-cofilin antibodies and found a polyclonal antibody provided by abcam (http://www.abcam.com/cofilin-antibody-ab42824.html) which I have added to the orders list on the wiki. This antibody is not conjugated to any fluorophores because we won't be using it for microscopy or flow cytometry. I think a polyclonal antibody is more suitable for what we're doing (abcam has a comparison of mono and polyclonal antibodies that I found useful http://www.abcam.com/index.html?pageconfig=resource&rid=11269&). I don't know how to evaluate what animal they were raised in though and what impact that has on our experiment so I'm going to look into that further tomorrow. The one I put on the orders page is a rabbit polyclonal antibody.
As to secondary antibodies, I am not really sure how to go about looking for/deciding on ones to use but Kathryn said you were going to provide some insight into that. 
Thanks for your help, Brian (and sorry for the wall of text..) 
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