Biocurious

/ a biophysics blog

Like Molasses on an Inclined Plane

Posted 22 March 2006 by Andre under

Things have been going slowly recently despite hours in the lab. That’s not to say I haven’t gotten anything done, but experiments that I was hoping would be easy are taking longer than they should (surprise!). On the bright side, I have discovered a new state of matter. I dub them ribo-rings. These glowing bastards may (or may not) be rhodamine labeled ribosomes that have self assembled into rings in order to mock me. I don’t know where they came from or where they went.

Although it should be clear that it’s sharp, no one ever tells you that the cutting edge hurts so much!

  

  1. PhilipJ    23 March 06    #

    What exactly is rhodamine labeling? There seems to be quite a high background of red, so is it a dye in the entire sample, or some site-specific labeling of the ribosomes?

    Keep us posted!


  2. Uncle Al    23 March 06    #

    90% of a project consumes 90% of the time allotted. The last 10% also consumes 90% of the time. If you are tightly managed, the last 1% of a project further consumes 90% of the time… and includes a PowerPoint presentation.

    Have you tried Alexa Fluor dyes or BODIPY fluors? The former have better output than rhodamine. The latter are less structurally perturbative (much smaller surface area, no net charge). They are especially good at longer wavelengths.

    As with all good things, the prices are high.


  3. Andre    23 March 06    #

    Phil,

    In this case we used a lysine reactive group to non-specifically label the ribosomes. The procedure basically involves throwing the label together with the ribosomes in a basic buffer and waiting. The unreacted dye is then separated from the ribosomes on a disposable column. This gave the ribosomes about 2 dyes each. The background is due to a few things: Maybe a little bit a free dye, labeled ribosomes that didn’t aggregate (the “signal”), and random fluorescent dirt on the slide (this particular slide was used right out of the box).

    Al,

    I haven’t tried other dyes yet, although of course I need to look into it. This data was collected under duress so I had to use whatever was available on short notice from a collaborator’s lab and this is what they had.

    It’s too bad that they are better at higher wavelengths because this is actually a combined AFM/fluorescence scope and it uses an 850 nm diode to detect the deflection of the cantilever and that light also needs to be filtered out (especially since the camera is very sensitive in the IR).


Name
Email
http://
Message
  Textile Help