This time, instead of having to soak the blot in one antibody, then another one, this blot I only had to soak in one antibody, as they've manage to get the marker to attach to the first antibody. This means that instead of having an antibody to your protein, (primary antibody) followed another antibody to your primary antibody (secondary antibody), you only need one, which cuts out half the steps.
This new one seems to have worked very well, now I've made up the various new solutions, because the method is different. I did have to put in 5 times as much developing solution as it said, but I think the solution I had was not quite as strong as the one on the sheet.
Image is of my blot. First column is a marker of proteins of known weight. Next two you can't see, as they are the negative control. Next two are pellet and supernatant from normal cells, next four are mutated cells (pellet, supernatant, P, S) and the last one is my protein on it's own.
This shows that my protein has stuck to the cell, but not all of it. I can tell that this is my protein, and not other proteins from the cell because my protein has a special marker on the end, called a His-tag (lots of the amino acid Histidine in a chain on the end), which my antibody can bind to.
This is the equivalent gel that I ran my proteins on. I did two gels with the same samples, one to blot and one to look at like this. These are with salt added to prevent charge reactions. You can see the marker on the left then the normal cells without my protein. You can see on the third column, there isn't a line, where there is on the 5th, 7th, 9th and 10th columns. The spread of bands on the other columns is the cell pellet, so this is all the contents from the cell. The other ones are the supernatant, which should just have my protein in. The very far right is just my protein on its own.
So, after impatiently waiting for my blot to develop, and then jumping around the lab because it had worked, I managed to calm down enough to learn how to do ITC. This stands for Isothermal Titration Calorimetry. Basically, you have two solutions of molecules, both in the same buffer, and you drop one into the other and see if there is any heat given off or taken in (heat exchange). If there is (a significant amount of) heat exchange then your two molecules bind.
There are a lot of steps to prepare the machine, but once you've done it, you can leave it to run, and get on with something else. The main thing (apparently) is to not have any bubbles or bits floating in your solution, both of which happen on a surprisingly regular basis. Managed to prepare my solutions, which took a bit of time, due to various different steps and needing to concentrate some of the proteins down.
Forgot how to use the nanodrop, which is a mini spectrophotometer (so it measures how much stuff you have in solution usually in grams per millilitre, by passing light through it and looking at scattering), but am now a pro, as I remembered the method I had of holding the end of the pipette like a pen, which works really well.
Whilst waiting for this to run, I grew some more cells up, as my supervisor realised that my proteins probably weren't binding to the bit of the cell I'm looking at, but rather an outer membrane protein (OmpF), already known to bind my protein. So for this set of cells, I have added the full length protein, mutated to not bind to OmpF, and a shortened protein that also can't bind OmpF.
Tomorrow, I will boil my samples to break up all the proteins so that they run properly on a gel; make the gels that I need; run the gels; run the blot; block the blot; wash the blot; add the antibody to the blot; wash the blot again and hopefully manage to develop the blot as well. I say hopefully, I have to do this, as I can't leave the blot at any stage for the whole weekend, otherwise it just won't work.
Will update on how all this goes next week. Need to start writing it up before long!
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