6th Week of Summer Research

Hi Readers,

In our cellular and developmental biology lab, we utilize different fixations to visualize the chromatin and proteins of interest. The two major fixations we utilize are an overnight methanol and 10 – 15 minute paraformaldehyde (aldehyde). Methanol fixations precipitate the proteins and preserve the structure of the proteins, but can cause denaturing of small soluble molecules. Paraformaldehyde cross links proteins by free amine groups, which ultimately forms intermolecular bridges (1).

Methanol and aldehyde fixations have been showing different SUMO patterns during anaphase I of meiosis I for C. elegans spermatogenesis: anaphase I is when the paired, homologous chromosomes are being separated, but the unpaired sex chromosome is “lagging.” The “lagging” unpaired sex chromosome is still present at the metaphase plate while the autosomes are being moved toward the spindle poles. In early anaphase I cells, methanol fixations have consistently been showing a diffuse pattern of SUMO associating with the chromatin, while aldehyde fixations are showing SUMO present in an intensely fluorescing ring complex during early anaphase I. I hypothesize the difference in patterns is due to the methanol fixation affecting the localization of the essential SUMOylation enzymes that must be present to preserve the ring complex structure integrity, while the aldehyde’s cross-linking nature is preserving the ring complex and depicting a more correct characterization of SUMO during early anaphase I.

I will further investigate the difference in SUMO patterns due to these different fixations by conducting more aldehyde preps and utilizing a shorter methanol exposure with acetone. Acetone will also precipitate the proteins, but is a milder solvent – I hope the acetone will better preserve the ring complex in contrast to the overnight methanol fixation.

Thank you for taking the time to read about my summer research! Please leave any questions you have in the comment section.

Katie McDonald

 

  1. Fixation and Permeabilization in ICC IF. (n.d.). Retrieved July 22, 2019, from https://www.novusbio.com/support/fixation-and-permeabilization-in-icc-if

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