Protein Sandwich

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Yesterday, I went to the lab to start a new western blot. 
With the same original Ba/F3 cells that were used for this IL3 withdrawal assay (my mentor thawed some cells from vials that I froze down before, so now there are a lot of healthy, fresh Ba/F3 cells in the incubator), I'm working on this next western blot. My mentor helped me lyse the cells with lysis buffer before I came, so that the cell lysate would be ready for me to begin the gel right after school. 

With lysates ready and sample buffer (with stinky BME) added to lysates, I brought the samples over to the western blot room. The proteins were denatured on a heat block. I prepared the gel box by filling it with the appropriate amount of SDS running buffer, getting the gels ready, pulling out the combs, etc. 

I did duplicates again (so two gels, one on each side of the box). I took the samples off the heat block, added loading dye, and loaded the wells. On each end of the gel I added the protein ladder (I learned from one of the research assistant IIs a smart way of knowing which order you put your samples in--load 10µl in lane 1, then 5µl after your last sample. The 10µl ladder will have thicker bands so then you know where you started. Otherwise you can remember that you can order your samples in alphabetical order, or make a chart)

After loading, I let the gel run. During that time, I went to count my cells from the IL3 withdrawal assay with 6% serum (Day 3). After that, I came back to do the transfer with my mentor. Basically, I'm transferring the invisible bands in the gel to a membrane so that when I probe them with antibodies and later with a chemiluminescent tag, the bands I'm interested in will light up. Last time, I probed with only anti NTRK1 and anti NTRK2 just to check for protein expression. Now, I'm going to probe with even more antibodies: a phospho antibody that looks for NTRK1 and NTRK2 phosphorylation. In addition, my mentor suggests that I should also probe ERK or MAP kinase, which I think is a good idea considering that NTRK is part of the MAPK pathway. 

Finally, for the transfer, I make this nice, sponge-wet filter paper-gel-methanol covered membrane-wetted filter paper-sponge sandwich. And then I dipped the whole thing in transfer buffer in the transfer tank/box and let it run overnight in the corner of the cold room. Yum. 

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Follow up questions

Does the order in which you probe the blot with these antibodies matter? Can you describe your plan for how you will interpret the presence/absence of ERK and MAP kinase in these cultures?