Zero Order


A research update!

Posted in Uncategorized by Amy Ross on December 27, 2010

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.

Zymography Gel

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.

 

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2 Responses to 'A research update!'

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  1. Kristin said,

    It’s interesting to read about your research Amy. I know this sounds silly, but did you compare the MMP from the cells to the MMP that (I assume) was purchased?? Also, is there any chance that the MMP produced by the cells isn’t being produced in a high enough quantity to be comparable to the MMP in PBS?? I don’t know where that takes your cell studies either… but it was cool to read this.

    Also, I hope you had a nice holiday 🙂 I am back to school and being swamped with studying soon myself.

    Best of luck with upcoming experiments 🙂

    • Amy Ross said,

      Hey Kristin:

      I was going to talk about this in a future post, but since you brought it up…

      Not only do I think it’s possible the cells aren’t producing enough MMP-2, I think it’s likely. Unfortunately, I can’t really prove it since zymography isn’t enitrely quantitative. I’ve observed there are brighter bands on the gel at higher cell densities. I’ve also tried using ConA to stimulate MMP activation, but it didn’t have an affect in the cells I’m using. Must keep trying things…


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