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MBJ Selenate Mass Spec (view source)
Revision as of 09:34, 25 September 2023
, 09:34, 25 September 2023→Agenda
=Agenda=
=Agenda=
07/15/2023: 10 rats (5 males, 5 females) were overdosed with euthasol and rapidly decapitated two hours after receiving 1ml/kg i.p. injections of 0.5mg/kg sodium selenate or vehicle (0.15M NaCl). Brains were dissected and washed in ice-cold PP2A storage buffer (Tris-HCl, pH 7.4, EDTA, EGTA, DTT, Sucrose, N-PER, protease inhibitor tablet [will update to include concentrations]), and frozen at -80*C.
'''07/15/2023 - Tissue Collection:
'''
10 rats (5 males, 5 females) were overdosed with euthasol and rapidly decapitated two hours after receiving 1ml/kg i.p. injections of 0.5mg/kg sodium selenate or vehicle (0.15M NaCl). Brains were dissected and washed in ice-cold 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), and frozen at -80*C.
{| class="wikitable"
{| class="wikitable"
| MBJ10 || F || Sel || 263 || 0.26 || 3:16 || 5:33
| MBJ10 || F || Sel || 263 || 0.26 || 3:16 || 5:33
|}
|}
'''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)
1:3 ratio of brain tissue:buffer
Dounce homogenization: 50-60 strokes on ice
The homogenate was centrifuged at 100,000 x g, 4°C for 1 hour
The supernatants were collected and stored at 4°C overnight
'''Sample clean-up: detergent and salt removal (7/29/2023)
'''
The supernatants were buffer exchanged into TEE buffer (50mM Tris-HCl pH 7.4, 0.1mM EDTA, 0.1mM EGTA) using PD-10 desalting columns (Cytiva #17085101)
The samples were then aliquoted and frozen at -20°C
'''Bradford and BCA assays to determine protein concentration (7/31/2023)
'''
Both Bradford and BCA Assays were conducted to determine the protein concentration. Results generated from both assays were nearly identical +/- 0.02ug/ml
The protein concentrations estimated from the BCA assay were used for all subsequent calculations
'''Reduction/Alkylation/Digestion (9/18/2023)
'''
Reduction
Add all 60 uL of each sample (see table 1 below) to a premade tube of 0.04g urea → [final] = 8M urea
Vortex until dissolved
Place in 37 deg C water bath for 2 mins
Add 5 uL TCEP bond breaker to each sample
pipette/mix 5x
Pulse 5 sec
Incubate in 37 deg C water bath for 60 mins
Alkylation
Add 10 uL of 20mM iodoacetamide stock solution to each sample
Vortex 2 seconds
Pulse 5 seconds
Incubate in 37 deg C water bath for 20 mins
Add 400 uL water to each sample → ~500 uL final volume
Digestion
Reconstitute 20ug Trypsin (Promega #V5111) by adding 200 ul of supplied buffer → [final] = 1ug/10ul trypsin
Add enough trypsin for a 1:20 ratio of trypsin:protein (see table 1 below)
Incubate all samples in 37°C water bath overnight
Next day:
Remove samples from water bath
Quench with 2.6 uL of 10% Formic Acid
Store at -20°C
'''Final sample
'''
Total number of samples = 17
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
Final volume = ~500uL per sample
=References=
=References=