45
edits
Changes
Jump to navigation
Jump to search
MBJ Selenate Mass Spec (view source)
Revision as of 10:39, 25 September 2023
, 10:39, 25 September 2023→Agenda: formatting again
Line 42:
Line 42:
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
Line 60:
Line 60:
'''''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
Line 88:
Line 88:
'''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=