Domain mapping of full-length huntingtin by limited proteolysis with chymotrypsin 2016/03/15
Preparing the full-length huntingtin Q17 (FL-HTT Q17) sample:
Protein kindly provided by Stefan Kochanek and Bin Huang (both of Ulm University, Germany – http://www.uniulm.de/gentherapie/en/site1.html), produced as detailed in their publication (http://www.ncbi.nlm.nih.gov/pubmed/25799558).
This sample is the same as that used in the previous limited proteolysis experiment: https://zenodo.org/record/45428.
Reminder: 100 μl aliquots purified protein at 300 μg/ml flash frozen in liquid nitrogen, equivalent to approximately 850 nM (the
MW of FL-HTT Q17 is 350 kDa). The protein sample buffer is 20 mM Tris, 500 mM NaCl, 0.4 % CHAPS, 5 mM DTT, 5 % glycerol,
pH 8.0. 2 x 100 μl aliquots shipped from Ulm to Toronto on dry ice and then stored at -80 °C. 2.5 μg/lane FL-HTT protein needed
for SDS-PAGE previously shown in preliminary experiments (completed by Bin Huang) to achieve visible bands on the gel by
coomassie staining.
1 aliquot thawed on ice and then centrifuged 20,000 xg, 4 °C, 1 min. No precipitate visible in tube before or after centrifugation.
Limited proteolysis reaction set up
7 limited proteolysis experiments set up with 15 μl FL-HTT Q17 protein (4.5 μg FL-HTT Q17) and 1 μl 9 μg/ml chymotrypsin stock
(9 ng chymotrypsin). This corresponds to a 1:500 (w/w) ratio of protease to target protein. Chymotrypsin stock prepared in same
buffer as FL-HTT Q17 (Sigma – C4129). 6 reactions incubated at 30 °C. Remaining reaction (time point 0 mins) immediately
quenched with 6 μl of 4x loading dye (Thermo Fisher Scientific NP0007) and incubating 95 °C, 2 mins. Reactions at 30 °C were
quenched in the same manner at time points of 5 mins, 10 mins, 20 mins, 30 mins, 60 mins and 180 mins.
SDS-PAGE of limited proteolysis reactions
Gel samples run using 10 well 4-20 % tris-glycine SDS-PAGE (Thermo Fisher Scientific XV04200PK20) using corresponding running
buffer (Thermo Fisher Scientific LC2675-4) at 200 V for 1 hour. Prestained protein ladders run either side of loaded samples
(Thermo Fisher 26619).
Staining of SDS-PAGE:
Same stock stain solution used as before and stirred throughout experiments to resuspend colloidal suspension.
Reminder: 100 g ammonium sulphate dissolved 800 ml ddH2O. 13.1 ml 85 % ortho-phosphoric acid added and mixed. 1 g
Brilliant Blue G-250 added and solution made up to 1 l total volume with ddH2O.
Gels removed from cassettes, rinsed in ultra-pure water then incubated in fixing solution (10 % acetic acid, 50 % methanol) for
10 mins. Gels rinsed in ultra-pure water then incubated in staining solution (80 % stain stock, 20 % methanol).
To avoid sample contamination for the subsequent stages of mass spectrometry analysis, throughout the handling of the gels, I
wore double gloves, a lab coat and ensured all equipment used was cleaned very thoroughly. Plasticisers can also contaminate
the mass spectrometry samples so the gel was washed, stained and destained in glass dishes.
Destaining of SDS-PAGE and visual inspection of limited digestion:
After staining for 2 hours, gels destained in ultra-pure water until background stain had sufficiently reduced and faint bands
were visible in the lanes.
Faint bands visible on gel, corresponding to cleaved domain fragments of HTT. Only 2 fragment bands are visible on the gel
around 100 kDa and 120 kDa, rather than the ladder of fragments seen with trypsin. The 100 kDa band seems to be
proteolytically stable beyond 1 hour of digestion, still faintly visible in the gel lane corresponding to the 3 hour digestion
reaction.
SDS-PAGE of FL-HTT Q17 digestion 1:500 (w/w) with chymotrypsin:
Bands indicated by the red arrows show proteolytically stable fragments ~ 100 kDa and 120 kDa
Next steps:
The gel was only stained for 2 hours and the bands are still very faint. I returned the gel to a fresh mix of stain to incubate
overnight in an effort to improve the contrast and visibility of the bands.