Debbie Knight

A day in the life: January 4, 2012

In research log on January 4, 2012 at 1:02 pm

New year and already things are off and running.

You know how some days you have one of those head-slap moments — a “how did I not think of that before” moments.

This week, the first of the year, I had one of those moments.

A few years ago I had been cranking out Western blots (a technique to look at proteins)* and all was going well. Then there was a three-year gap because I was working in another lab. Upon my return to my “old” lab, I started up the ol’ Western blotting operation again — only this time things did not go so smoothly.

The numerous protein bands I had come to expect on the blots just weren’t there. Increasing the amount of protein I added to each sample didn’t help. Making new buffers and new formulations of those buffers didn’t help. Using new reagents to detect those proteins didn’t help.

It wasn’t until I ran out of the nitrocellulose membrane – the paper-like substance the proteins were stuck on – and I opened a new box of membrane that it became obvious what the problem was. The membrane stock I had been using was over four or five years old. Things had happened to that membrane – things like exposure to air and temperature fluctuations – caused the little charges that helped hold the proteins onto the membrane to change in some way and loose their ability to grip the proteins.

This was something I so obviously should have considered when I was trying to figure out why the procedure wasn’t working like it had a few years before.

(D’oh!)

A side-by-side comparison of old and new stocks of nitrocellulose membrane (a paper-like material the hold on to proteins during a laboratory technique called a Western blot). There are more protein bands detected on the new membrane and the proteins are "crisper" compared to those on the old membrane. The Western blots were run on different days for different times which is why the protein bands do not exactly line up with each other even though they are from the same sample. The lines between the two images show where the corresponding protein bands are located.

The proteins are barely detectable on the old membrane.

So, as you can see by the photos I’ve included in this post, there are more protein bands and they are “crispier” on the new membrane than on the old membrane.

I guess, after 20 years in the lab, I still have a few things left to learn.

Go figure!

*If you would like to see how a Western blot is done, see my previous post.

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  1. I think about all the time that’s lost with moments like this–expired products that you don’t know are expired, such as membranes, reagents, antibodies, etc. Congrats on figuring out the cause, a scientist you are!

    On a side note, when purchasing new membranes, ask your sales rep for a discount. Those profit margins are usually marked up for NC membranes (among many other products).

  2. Lol! So true. 20 years in the lab and I still learn new things all the time! As an instructor for upper level lab classes, I’d also be continually surprised in how students can find new and accidentally innovative ways to completely screw up a protocol! Quick way to learn every lab mistake that is possible to make!

    I am paying “blog calls” to each @scio12 attendee to say “Hi” and give your blog a shoutout on twitter (I’m @sciencegoddess). I look forward to meeting you in January!

    • Joanne: I was one of those students once. As for my experiments in the lab, I thought I had covered all the possible things that could go wrong with a protocol (by the ol’ trial & error method, of course), but there’s always something. See you @scio12 !

  3. Mmmm… As a marketer working for Novex, part of Life Technologies I so want to say something clever in response to Tom’s comment…

    OK, here it is:

    (silence)

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