The sample tested was a new compound (described in previous post), which I had synthesized the day before. Part of the "triazolopyrimidine" family of compounds, it is structurally similar to previously tested compounds. We tested the compound for its "MT (microtubule) stabilizing" ability. A common symptom in Alzheimer's patients is the destabilization of neuronal cell microtubules. This leads to cell death, as the microtubule plays an important role in intracellular transport. Thus, compounds with the "MT stabilizing" properties are at the focus of Alzheimer's research.
When I got to the lab in the morning, I met Jane. Jane had been a post-doc at the lab for a year and a half, and had done her graduate work at Temple on cocaine drug studies. The first thing she did was show me around the lab, which was a pretty big surprise to me. The lab was only 3 years old, compared to our 42 year old chemistry lab, with a beautiful view of the campus and spacious, clean work spaces Despite my love for our chemistry lab, I had to admit that the medical lab seemed much nicer, an opinion that my other lab-mates agreed with.
While Jane showed me around the lab, she explained the steps of the experiment. In her words, we were to "test the microtubule stabilizing" effects of the compound on "QBI-293" HEK kidney cells through an "ELIZA test". In order to do that though, we would first have to conduct a "BCA protein assay" in order to determine the concentrations of the proteins in advance. I did not understand anything she was talking about, until she explained it to me in laymen terms.
"So basically", she said, "we're adding the compound to the kidney cells, then using a lysis buffer to open the membrane and collect the proteins."
(Wait...so the cells die in the process)
"Yeah, the cells die when you add the buffer. That way it prevents degrading and accumulation of protein."
"Then we'll add BSA to the cells, which will help change the color of the cells for the BCA protein assay.
(Wait, what's BSA).
"Bovine Serum Albumin. Write that down. It's important for determining protein concentration."
(Got it).
"So, using a BCA protein assay, we can find the concentrations of the proteins. Basically, depending on the concentrations of protein, the cells will change color. So by measuring the color change of the cells, we can then quantitatively determine their protein concentration."
(And that's it?)
"No, that's actually the preparation for the ELIZA test itself. We need to know the concentrations of protein to conduct the ELIZA. You'll have to come in tomorrow to finish that."
So, for the rest of the first afternoon, I shadowed Jane as she conducted the preparation for the ELIZA test. It being my first time in a biology lab, I had a lot of questions for her, which she answered patiently. There was a lot of precise pipet work and centrifuging involved, but I was just waiting a large portion of the time. I was definitely surprised by how much down time there was between the different tests and assays. (In the chemistry lab, there are often several different reactions and purifications occurring at once, meaning that we're at work more frequently).
So the next day, I showed up early for the ELIZA test.
"So today, we'll be conducting the ELIZA test."(Alright).
"By any chance, do you know what it might stand for?"(No, I'm not really sure).
"Alright, thats fine. It stands for Enzyme-Linked immunosorbent assay. What we've doing is we're adding capture and reporter antibodies to the kidney cells from before. These antibodies will bind to two types of microtubulin, and have HRP "tags" that will change color depending on the protein concentration."(So kind of like the BSA?)
"Yeah"(Alright, and why is this important?)
"Well, that's what this experiment is all about. This color change allow us to detect the concentrations of two substances: acetylated tubulin and alpha tubulin. And after we collect this quantitative data, we can use it to see the microtubulin levels, to see how effective a stabilizer the compound is".(Ahhh).(By the way, what does HRP stand for?)
"It stands for horseradish peroxidase. You might want to write that down".
Similar to the BCA assay from before, the ELIZA test worked based on color change and concentration. The two different antibodies changed color based on tubulin concentration, thanks to the HRP tag; this meant that by measuring this color change, using a specialized machine, we could quantify the microtubulin levels.
To sum the entire test up, we added the compound to the cells, first checked the protein levels using the BCA assay (which was needed to set up the ELIZA test), then used the ELIZA test to find the final concentration of microbutulin present in the cells. Gathering this data then, we were able to determine whether the compound was a potent microtubulin stabilizer based on the level of acetylated microtubulin present and whether the compound was harmful towards microtubulin levels in general (which is a negative characteristics), based on alpha tubulin levels.
When we finally performed the statistical analysis on the data (Dunnett Test, Nova test), we were able to see that the cells treated with the compound had a statistically significant increase in levels of acetylated tubulin (indicating strong microtubulin stabilizing properties) and no decrease in levels of alpha tubulin (indicating that it is not harmful to tubulin in general), meaning that the compound shows promise for further testing.
When I got to the lab in the morning, I met Jane. Jane had been a post-doc at the lab for a year and a half, and had done her graduate work at Temple on cocaine drug studies. The first thing she did was show me around the lab, which was a pretty big surprise to me. The lab was only 3 years old, compared to our 42 year old chemistry lab, with a beautiful view of the campus and spacious, clean work spaces Despite my love for our chemistry lab, I had to admit that the medical lab seemed much nicer, an opinion that my other lab-mates agreed with.
While Jane showed me around the lab, she explained the steps of the experiment. In her words, we were to "test the microtubule stabilizing" effects of the compound on "QBI-293" HEK kidney cells through an "ELIZA test". In order to do that though, we would first have to conduct a "BCA protein assay" in order to determine the concentrations of the proteins in advance. I did not understand anything she was talking about, until she explained it to me in laymen terms.
"So basically", she said, "we're adding the compound to the kidney cells, then using a lysis buffer to open the membrane and collect the proteins."
(Wait...so the cells die in the process)
"Yeah, the cells die when you add the buffer. That way it prevents degrading and accumulation of protein."
"Then we'll add BSA to the cells, which will help change the color of the cells for the BCA protein assay.
(Wait, what's BSA).
"Bovine Serum Albumin. Write that down. It's important for determining protein concentration."
(Got it).
"So, using a BCA protein assay, we can find the concentrations of the proteins. Basically, depending on the concentrations of protein, the cells will change color. So by measuring the color change of the cells, we can then quantitatively determine their protein concentration."
(And that's it?)
"No, that's actually the preparation for the ELIZA test itself. We need to know the concentrations of protein to conduct the ELIZA. You'll have to come in tomorrow to finish that."
So, for the rest of the first afternoon, I shadowed Jane as she conducted the preparation for the ELIZA test. It being my first time in a biology lab, I had a lot of questions for her, which she answered patiently. There was a lot of precise pipet work and centrifuging involved, but I was just waiting a large portion of the time. I was definitely surprised by how much down time there was between the different tests and assays. (In the chemistry lab, there are often several different reactions and purifications occurring at once, meaning that we're at work more frequently).
So the next day, I showed up early for the ELIZA test.
"So today, we'll be conducting the ELIZA test."(Alright).
"By any chance, do you know what it might stand for?"(No, I'm not really sure).
"Alright, thats fine. It stands for Enzyme-Linked immunosorbent assay. What we've doing is we're adding capture and reporter antibodies to the kidney cells from before. These antibodies will bind to two types of microtubulin, and have HRP "tags" that will change color depending on the protein concentration."(So kind of like the BSA?)
"Yeah"(Alright, and why is this important?)
"Well, that's what this experiment is all about. This color change allow us to detect the concentrations of two substances: acetylated tubulin and alpha tubulin. And after we collect this quantitative data, we can use it to see the microtubulin levels, to see how effective a stabilizer the compound is".(Ahhh).(By the way, what does HRP stand for?)
"It stands for horseradish peroxidase. You might want to write that down".
Similar to the BCA assay from before, the ELIZA test worked based on color change and concentration. The two different antibodies changed color based on tubulin concentration, thanks to the HRP tag; this meant that by measuring this color change, using a specialized machine, we could quantify the microtubulin levels.
To sum the entire test up, we added the compound to the cells, first checked the protein levels using the BCA assay (which was needed to set up the ELIZA test), then used the ELIZA test to find the final concentration of microbutulin present in the cells. Gathering this data then, we were able to determine whether the compound was a potent microtubulin stabilizer based on the level of acetylated microtubulin present and whether the compound was harmful towards microtubulin levels in general (which is a negative characteristics), based on alpha tubulin levels.
When we finally performed the statistical analysis on the data (Dunnett Test, Nova test), we were able to see that the cells treated with the compound had a statistically significant increase in levels of acetylated tubulin (indicating strong microtubulin stabilizing properties) and no decrease in levels of alpha tubulin (indicating that it is not harmful to tubulin in general), meaning that the compound shows promise for further testing.
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