Preparing Cell Lysates: Difference between revisions

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Line 5:

! || Final Concentration || per 10 mL || Stock

|-

|Tris pH7.4 || 50mM || 500uL || 1M

|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)

|-

|Na Deoxycholate || 0.25% || 250uL || 10%

Line 15:

|EDTA || 1mM || 20uL || 0.5M

|-

|NaVO3 || ~~1mM~~ || ~~100uL~~ || 1M

|NaVO3 (see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM

|-

|NaF || 5mM || 100uL || ~~250mM~~

|NaF || 5mM || 100uL || 0.5M

|-

|NaPPi || 25 mM || 1 mL || ~~500mM~~

|NaPPi || 25 mM || 1 mL || 250mM

|-

|- Protease Inhibitors !! 1 tab

|Protease Inhibitors || || 1 mini tab ||

|}

==Basic Protocol==

# Stimulate cells if necessary

# Stimulate cells if necessary (i.e. insulin treatment for 10 min)

# Wash cells 2x1mL with ice cold PBS -/- and aspirate

# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate

# Add 200uL [[Buffer~~/RIPA|~~RIPA]] buffer and scrape cells

# Add 200uL Buffer (RIPA buffer) and scrape cells

# Pipet into cold eppendorf tubes

# rotate end over end for 30 minutes at 4C to lyse

# rotate end over end for 30 minutes at 4oC to lyse

# Centrifuge 10 min at 13,000 RPM to clarify

# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS

# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS

# Load gel

@@ Line 5: / Line 5: @@
 ! || Final Concentration || per 10 mL || Stock
 |-
-|Tris pH7.4 || 50mM || 500uL || 1M
+|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
 |-
 |Na Deoxycholate || 0.25% || 250uL || 10%
@@ Line 15: / Line 15: @@
 |EDTA || 1mM || 20uL || 0.5M
 |-
-|NaVO3 || 1mM || 100uL || 1M
+|NaVO3 (see [[Preparation_of_Orthovanadate_Stocks | preparation ]]) || 100uM || 10uL || 100mM
 |-
-|NaF || 5mM || 100uL || 250mM
+|NaF || 5mM || 100uL || 0.5M
 |-
-|NaPPi || 25 mM || 1 mL || 500mM
+|NaPPi || 25 mM || 1 mL || 250mM
 |-
-|- Protease Inhibitors !! 1 tab
+|Protease Inhibitors || || 1 mini tab ||
 |}
 ==Basic Protocol==
-# Stimulate cells if necessary
+# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
-# Wash cells 2x1mL with ice cold PBS -/- and aspirate
+# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
-# Add 200uL [[Buffer/RIPA|RIPA]] buffer and scrape cells
+# Add 200uL Buffer (RIPA buffer) and scrape cells
 # Pipet into cold eppendorf tubes
-# rotate end over end for 30 minutes at 4C to lyse
+# rotate end over end for 30 minutes at 4oC to lyse
 # Centrifuge 10 min at 13,000 RPM to clarify
-# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
+# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
 # Load gel