Drummond:Protein Isolation: Difference between revisions
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== Protocol to isolate total, soluble and insoluble protein fractions from | == Protocol to isolate total, soluble and insoluble protein fractions from isotopically labeled strains of ''S. cerevisiae'' == | ||
===Day -2=== | |||
* For each strain, inoculate a 3 mL YPD pre-culture of YPD with a single yeast colony and incubate overnight at 30⁰C with shaking or rotation. | |||
* Inoculate | ===Day -1=== | ||
* Inoculate 50 mL of synthetic complete medium (SC) with labeled amino acids with 3 µL of the overnight pre-culture and incubate for 18 h at 30⁰ C with shaking in a 500 mL baffled flask. | |||
===Day 0=== | |||
* Measure the optical density of the 50 mL assay cultures to confirm that it is at an OD<sub>600</sub> of around 0.400. Transfer 45 mL of the assay culture to a new 500 mL baffled flask and induce with 40 mM galactose (add 1622 µL of 20% galactose) for 2 h at 30⁰ C with shaking. Continue to incubate the original flask of remaining uninduced cells alongside the flask of galactose-induced cells. | |||
* | |||
== Buffer recipes == | == Buffer recipes == | ||
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* 45.625 mL protease-free H<sub>2</sub>O | * 45.625 mL protease-free H<sub>2</sub>O | ||
Then mix 9.8 mL of WB stock solution with 100 µL 100mM PMSF and | Then mix 9.8 mL of WB stock solution with 100 µL 100mM PMSF and 100µL protease inhibitors to make 10mL WB. | ||
=== Insoluble Protein Buffer, pH 8.5 (IPB) === | === Insoluble Protein Buffer, pH 8.5 (IPB) === | ||
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Then mix 475 µL of 6x SPLB stock solution with 25 µL 14.3M βME immediately before use. | Then mix 475 µL of 6x SPLB stock solution with 25 µL 14.3M βME immediately before use. | ||
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Revision as of 08:29, 6 April 2012
Protocol to isolate total, soluble and insoluble protein fractions from isotopically labeled strains of S. cerevisiae
Day -2
- For each strain, inoculate a 3 mL YPD pre-culture of YPD with a single yeast colony and incubate overnight at 30⁰C with shaking or rotation.
Day -1
- Inoculate 50 mL of synthetic complete medium (SC) with labeled amino acids with 3 µL of the overnight pre-culture and incubate for 18 h at 30⁰ C with shaking in a 500 mL baffled flask.
Day 0
- Measure the optical density of the 50 mL assay cultures to confirm that it is at an OD600 of around 0.400. Transfer 45 mL of the assay culture to a new 500 mL baffled flask and induce with 40 mM galactose (add 1622 µL of 20% galactose) for 2 h at 30⁰ C with shaking. Continue to incubate the original flask of remaining uninduced cells alongside the flask of galactose-induced cells.
Buffer recipes
Stock solutions
- 200mM DTT
- 100mM PMSF
- 4M NaCl
- 1M Tris-HCl, pH 8.5
- 20% SDS (w/v)
Soluble Protein Buffer, pH 8.5 (SPB)
(50mM Tris-HCl, 500mM NaCl, 1:100 protease inhibitors)
Start by making 50 mL SPB stock solution:
- 2.5 mL 1M Tris-HCl, pH 8.5
- 6.25 mL 4M NaCl
- 41.25 mL protease-free H2O
Then mix 9.8 mL of SPB stock solution with 100 µL 100mM PMSF and 100uL protease inhibitors to make 10mL SPB.
For protease inhibitors, we use:
- AEBSF, HCl, MW=239.5, targets serine proteases, 100 mM
- E-64, MW=357.4, targets cysteine proteases, 1.5 mM
- Pepstatin A, MW=685.9, targets aspartic proteases, 2 mM
- o-Phenanthroline, MW=198.2, targets metalloproteases, 500 mM
This mixture is commercially available as Calbiochem protease inhibitor cocktail set IV, or BioVision EZBlock protease inhibitor cocktail IV.
Wash Buffer (WB)
(50mM Tris-HCl, 150mM NaCl, 1:100 protease inhibitors) Same as SPB, but with reduced salt to limit pellet loss.
Start by making 50 mL WB stock solution:
- 2.5 mL 1M Tris-HCl, pH 8.5
- 1.875 mL 4M NaCl
- 45.625 mL protease-free H2O
Then mix 9.8 mL of WB stock solution with 100 µL 100mM PMSF and 100µL protease inhibitors to make 10mL WB.
Insoluble Protein Buffer, pH 8.5 (IPB)
(50mM Tris-HCl, 150mM NaCl, 8M urea, 2% SDS, 1mM PMSF, 2mM DTT, 1:1000 protease inhibitors) Make fresh before each use. Note that at 8M, urea occupies ~72% of the solution volume, so that 4.8g urea occupies 7.2mL of a 10mL solution. Do not use 500mM NaCl in this solution, as it loosens pellets and causes sample loss.
- 8M urea
- 2% SDS
- 50mM Tris pH 8.5
- 150mM NaCl
- 1mM PMSF
- 20 μL 200mM DTT
- 1:1000 protease inhibitors (see above. higher concentrations may crash out.)
Use 500μL IPB per sample, and note that foaming will make ~25% of the solution unusable.
2mL IPB (~2 samples) | 13mL IPB (~10 samples) |
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6x Soluble Protein Loading Buffer, pH 6.8 (SPLB)
(50 mM Tris-HCl pH 8.5, 10% SDS, 0.05% bromophenol blue, 30% glycerol, 5% β-mercaptoethanol [βME])
Start by making 9 mL 6x SPLB Stock Solution:
- 0.5 mL 1M Tris-HCl, pH 8.5
- 1 g SDS
- 5 mg bromophenol blue
- 3 mL glycerol
- 5.5 mL protease-free water
Then mix 712.5 µL of 6x SPLB stock solution with 37.5 µL of 14.3M βME immediately before use.
6x Insoluble Protein Loading Buffer, pH 6.8 (IPLB)
(50 mM Tris-HCl, 0.05% bromophenol blue, 30% glycerol, 5% βME)
Start by making 9 mL 6x SPLB Stock Solution:
- 0.5 mL 1M Tris-HCl, pH 6.8
- 5 mg bromophenol blue
- 3 mL glycerol
- 5.5 mL protease-free water
Then mix 475 µL of 6x SPLB stock solution with 25 µL 14.3M βME immediately before use.