I’ve still been working on MMP-2 mediated peptide release from hydrogels. I did another study with just MMP-2 in buffered saline, and it worked nicely. The MMP group had much greater peptide release than the non-MMP group. So why was it, when I used MMP-2 producing cells, that there was no difference in the group with cells and the control group?
One hypothesis I had was that the cells were either a) not producing MMP-2, or b)not producing it in the presence of hydrogels. To test this, I had to learn a technique called gelatin zymography. It’s similar to SDS-PAGE, gel electrophoresis technique is to separate and identify proteins. Here’s how it works.
Gel Electrophoresis in Two Minutes
If you want to identify your sample has a specific protein, gel electrophoresis can help you identify it.* Samples (and controls) are put in a buffer with sodium dodecyl sulfate (SDS) and dye. The SDS partially denatures the protein (unfolds it, thereby rendering it nonfunctioning, but easier to move through the gel!) and gives it a negative charge. All the samples are loaded into a polyacrylamide gel. The gel is placed in an electric field, with the negative charge at the loading end, and a positive charge at the opposite end. Now negatively charged, the proteins are attracted to the positive end and start travelling through the gel.
However, the smaller the protein is, the faster it can travel through the gel. Threfore, the proteins sort themselves by size in each sample, with the smallest being towards the bottom, and the largest towards the top. The dye in the buffer is used to track how far the sample has moved. When the dye is most of the distance through the gel,it is removed from the electric field and stained. The proteins show up as bands on the gel.
How is Gelatin Zymography Different?
In a zymography gel, an enzyme substrate (in this case, gelatin) is added. After removing from the electric field, the gel is placed in a renaturing solution, to let any present enzymes refold into their functional conformations. The gel is incubated at 37 degrees C, which allows the enzymes to degrade any substrate in their vicinity. Then the gel stained. Unlike an SDS-PAGE, since protein is present throughout, the whole gel stains and the bands are clear. The presence of enzyme is indicated by a lack of protein.The downside to this technique is it’s not quantititative. You can confirm the presence or absence of active enzyme, but you can’t translate it to an amount of protein or a concentration. There are other techniques to do this for proteins such as ELISA or a Western Blot, but they can’t be used for MMP-2, for reasons I’m not going to go into here.
What were my results?
Well, it took a few tries to learn this technique. Everyone had to help me with some aspect of it, even our visiting high school student! But after 5 or 6 gels, I felt like I had the hang of it. I have confirmed:
-The cells I’m using are producing active MMP-2
-The cells produce active MMP-2 in the presence of hydrogels
So what was the next step for my cell studies? I’ll leave that for next time.
*Gel electrophoresis is also used to identify DNA segments. Since DNA is negatively charged already, there’s no need to add SDS. DNA gels are done in a material called agarose.
After three months of not posting, I’m back. For a while, I didn’t have a lot I could talk about, and I wasn’t feeling motivated. But I want to keep blogging. I want to keep writing about what I’m working on.
Speaking of writing…I have the potential for middle authorship on a scientific paper!
First, a little intro about scientific authorship. It’s expected that all grad students publish at least one paper while they are in school. Generally speaking, multiple publications are better, especially if you plan to have an academic career. The first author on any paper is the person whose project it is, and did the majority of the work and writing. The last author is the senior author, i.e., the PI. Authorship can be obtained by the contribution of ideas, writing, or data interpretation.
I didn’t expect to have any more than the one paper I will write on my thesis project. However, a former lab member just had a manuscript rejected by a journal, and my PI asked me, along with another member of our lab, if we’d like to rewrite portions of it.
It would be awesome to be listed as a second or third author,but I feel kinda intimidated by this. My PI has witheld many of his comments about the reviewers’ criticisms, because he doesn’t want to “unduly influence” us. In some ways, it’s like “You want to know what I think about how to improve this paper? I’ve never written a journal article.” There were several things I noticed that caught my attention, but initially dismissed. For instance, when looking at the data, I thought there needed to be measurements taken at more time points, but didn’t give it further thought. When I saw the reviewers’ comments, they said the exact same thing.
Despite my intimidation, I’ve really gotten into the project. I read through the thesis the work is based on (all 191 pages), and I’ve outlined a number of improvements. My main criticism is that the introduction and the language need to be more concise. I’ll keep working on it, and we’ll see if it gets published.