Changes

For each frozen tissue sample (MBJ01-10), Amygdalar lobe (A) and caudal brainstem (B) regions were dissected and homogenized in PP2A storage buffer (50mM Tris-HCl, pH 7.4, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 250mM Sucrose, 0.1%(v/v) N-PER detergent, 1 Pierce protease inhibitor mini tablet per 10mL solution)

For each frozen tissue sample (MBJ01-10), Amygdalar lobe (A) and caudal brainstem (B) regions were dissected and homogenized in PP2A storage buffer (50mM Tris-HCl, pH 7.4, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 250mM Sucrose, 0.1%(v/v) N-PER detergent, 1 Pierce protease inhibitor mini tablet per 10mL solution)

1:3 ratio of brain tissue:buffer

1:3 ratio of brain tissue:buffer

Dounce homogenization: 50-60 strokes on ice

* Dounce homogenization: 50-60 strokes on ice  

The homogenate was centrifuged at 100,000 x g, 4°C for 1 hour

* The homogenate was centrifuged at 100,000 x g, 4°C for 1 hour  

The supernatants were collected and stored at 4°C overnight  

* The supernatants were collected and stored at 4°C overnight  

'''7/29/2023 - Sample clean-up: detergent and salt removal  

'''7/29/2023 - Sample clean-up: detergent and salt removal  

'''''Reduction'''''

'''''Reduction'''''

* Add all 60 uL of each sample (see table 1 below) to a premade tube of 0.04g urea → [final] = 8M urea

* Add all 60 uL of each sample to a premade tube of 0.04g urea → [final] = 8M urea

* Vortex until dissolved

* Vortex until dissolved

* Place in 37 deg C water bath for 2 mins

* Place in 37 deg C water bath for 2 mins

'''Final sample  

'''Final sample  

'''

'''

Total number of samples = 17

* Total number of samples = 17

09A, 09B, and 10B were excluded from this batch due to possible cross-contamination

* 09A, 09B, and 10B were excluded from this batch due to possible cross-contamination

Buffer (before reduction/alkylation/digestion) = 50mM Tris-HCl pH 7.4, 0.1mM EDTA, 0.1mM EGTA

* Buffer (before reduction/alkylation/digestion) = 50mM Tris-HCl pH 7.4, 0.1mM EDTA, 0.1mM EGTA

Final volume = ~500uL per sample

* Final volume = ~500uL per sample

=References=

=References=