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Line 46: − + − + − + − + − + − + − + + + + + − + − 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 − − − − + − + − +
MBJ Selenate Mass Spec (view source)
Revision as of 09:37, 25 September 2023
, 09:37, 25 September 2023→Agenda: formatting
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