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MBJ Selenate Mass Spec (view source)
Revision as of 10:37, 25 September 2023
, 10:37, 25 September 2023→Agenda: formatting
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pipette/mix 5x+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
−
The supernatants were collected and stored at 4°C overnight
The supernatants were collected and stored at 4°C overnight
−'''Sample clean-up: detergent and salt removal (7/29/2023)
+'''7/29/2023 - Sample clean-up: detergent and salt removal
'''
'''
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 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
The samples were then aliquoted and frozen at -20°C
−'''Bradford and BCA assays to determine protein concentration (7/31/2023)
+'''7/31/2023 - Bradford and BCA assays to determine protein concentration
'''
'''
Both Bradford and BCA Assays were conducted to determine the protein concentration. Results generated from both assays were nearly identical +/- 0.02ug/ml
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
The protein concentrations estimated from the BCA assay were used for all subsequent calculations
−'''Reduction/Alkylation/Digestion (9/18/2023)
+'''9/18/2023 - Reduction/Alkylation/Digestion
'''
'''
−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 (see table 1 below) 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
+* 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
−Add 5 uL TCEP bond breaker to each sample
+'''''Alkylation'''
−''
−Pulse 5 sec
+* Add 10 uL of 20mM iodoacetamide stock solution to each sample
−Incubate in 37 deg C water bath for 60 mins
+* 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
−* Incubate all samples in 37°C water bath overnight
−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:
Next day:
−Remove samples from water bath
+* Remove samples from water bath
−Quench with 2.6 uL of 10% Formic Acid
+* Quench with 2.6 uL of 10% Formic Acid
−Store at -20°C
+* Store at -20°C
'''Final sample
'''Final sample