Changes

'''7/28/2023 - Protein Extraction:'''

'''7/28/2023 - Protein Extraction:'''

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  

'''''Reduction'''''

'''''Reduction'''''

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

* Add 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