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MBJ Selenate Mass Spec (view source)
Revision as of 10:34, 25 September 2023
, 10:34, 25 September 2023→Agenda
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=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"
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| 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=