Zero Order


Troubleshooting fun!

Posted in Uncategorized by Amy Ross on January 24, 2011

Did something pretty stupid in lab la week. A few weeks ago I talked about gel zymography. I had some initial difficulties learning the technique, but I feel reasonably competent now that I’ve done it enough times. Or so I thought.

I keep all my buffers and solutions on a shelf above my bench. To run a gel, you fill the electrophoresis cell (a plastic box that holds the gel and apparatus) with running buffer. So I pulled the jar of running buffer off the shelf, on to my bench, and filled the cell. Then I closed the cell, applied the electrical field, and let it run. Normally, I see well-defined blue bands travel down the gel as it progresses. But  the bands were blurry, faded, and the ladder (prestained protein markers) was smudged  and bent. I’d had some issues with gels with “frowny” bands about a month ago. I had done some Googling and concluded the problem was my sample buffer had gotten old. I made new sample buffer and resumed running gels without issue.  So I didn’t think that was the problem this time.

Then I looked at the bottle on my bench.

It wasn’t running buffer.  Crap.

Things I did wrong:

-I have no distinct labeling system for my bottles. I use purple tape for everything. I should do different colors for solutions that have no other distinguishing characteristics (i.e., color).  I might switch to different colors, or use a numbering system like hair dye.

-Obviously, I didn’t read the label before pouring. Kind of important.

Things I did right:

-I figured out what was causing the problem.

-I didn’t throw away my leftover samples, making it easier to set up another gel.

-I used a ladder.  The ladders haven’t been showing up when I photograph the gels. I’m only looking at one molecular weight, and I’ve never had a problem with the MMP-2 bands showing up in a range other than between the 50 and 85 kDa bands on the ladder (active MMP-2 has  a molecular weight of 66-72 kDa, so the bands are showing up in the expected position). Despite this,  I’ve continued using one every time.  In this case, it was valuable as an indicator that something was wrong. It only takes a few minutes to defrost and inject the solution, and ladders aren’t expensive. I think the benefits outweigh the costs.

I developed and stained the failed gel just to see how it turned out. The bands were present, but bent. The bands were also higher than their normal position, but I attribute that to my stopping the run when I realized my mistake. The run time was about 45 minutes, rather than the usual 60-75 minutes.

Failed gel post staining and destaining.

If you’re interested, I found the SDS-PAGE Hall of Shame helpful and amusing. I may set up my own “Hall of Shame” to help future lab members troubleshoot their gels!

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An update on MMP production

Posted in Uncategorized by Amy Ross on January 16, 2011

Soooooo..in a comment to my last entry, I remarked that not only was it possible my cells weren’t producing enough active MMP-2 to produce a measurable release of peptide in the hydrogels, I thought it was likely. It’s a hypothesis I’ve been pursuing for about a month. As we’ve talked about before, cell lines in culture don’t always behave the same way as their primary cell counterparts in vivo. They’re different genetically, they don’t produce MMP while living on tissue culture plastic…there could be any number of reasons.

I started looking at papers on MMP-2 expression, and how other groups boosted theirs. One option is to use chemicals to convert the inactive form of the enzyme to the active form. I found one paper that used plates coated in extracellular matrix proteins boosted their MMP-2 presence.

This was a fairly easy hypothesis to test. I ran two experiments. In one, I did an in-vitro peptide release study, and saved the media at each time point.  In the second, I collected media after 48 hours of exposures to my cells, and exposed hydrogels to this media (hereafter referred to as conditioned media). I ran gel zymography on the samples from the in vitro study and the conditioned media study.

The results? Unsupportive of my hypothesis. The gel showed plenty of active MMP-2 production by the 48 hour mark in the in vitro study and in the conditioned media study. However, there was no significant difference in peptide release compared to the controls (in both cases, hydrogels in media only).

Sigh.

Next hypothesis: the MMP-2 may be there, but is it active? Testing this at the moment with mixed results. Hopefully I”ll have more to talk about later!

In other news, my wonderful husband gave me an iPad for Christmas. I’m thinking of downloading the Keynote app and doing my group meeting presentations from the iPad. One of our rotation students from last semester did some nice work with Keynote. I think incorporating some more of the sophisticated animations could be useful without being obnoxious.