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[[ Category: Protein Purification ]]
[[ Category: Protein-Protein Interactions ]]
[[ Category: Protein Biochemistry ]]
[[ Category: Proteins ]]
__NOTOC__
==Buffers==
Prepare each of these and chill on ice:
* Nucleotide Stripping Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 20 uL 0.5M EDTA
* Loading Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 100 uL 1M MgCl
* Lysis Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 4.5 mL water + 500 uL 20% Triton X100 + PI Tablet
* Wash Buffer: 25 mL [[ 2xHNG_Buffer | 2x HNG ]] + 25 mL water
==Preparation of Immobilized GTPases==
#Make 20 ug aliquots of GTPases immobilized to glutathione beads (see [[ GST Pulldown Assay ]])
#Resuspend one aliquot in 1 mL Nucleotide Stripping Buffer
#Incubate at Room Temperature for ~ 20min
#Spin down beads, aspirate supernatant and wash 1 x with 1 mL Loading Buffer.
#Aspirate supernatant and add 500 uL Loading Buffer plus 10 uL GDP or GTPyS
#Incubate at room temperature for about 20 min
==Preparation of Lysates==
#Wash cells 2x with 25 mL PBS (for 150 mm plate; use 10 mL for 100 mm dish)
#Add 2 mL Lysis buffer (or 1 mL for 100 mm dish) and scrape cells using plastic spatula. Collect in 1.5 mL eppendorf tubes
#Incubate end over end for 20 min at 4 C
#Centrifuge 10 min at 13 000 RPM at 4C to clarify
#Remove supernatants to fresh tubes.
#Save a lysate sample in 5X SDS-Cocktail
==Pull-Down Assay==
#Add appropriate amount of lysate to nucleotide-loaded beads. Typically we use 2 ug for a pull-down, so that corresponds to 1/5 of the resuspended GST-GTPase solution. You can vary the beads and the lysate but a good starting point is 500 ug protein lysate. If necessary bring volume of assay up to ~1 mL with HNG
#Add 50 uL of Glutathione Beads for bulk during washing.
#Incubate end over end at 4C for 30 min to 1h.
#Wash beads 5 times with 1 mL Wash Buffer
#Resuspend beads in 50 uL of 5X SDS Cocktail
#For analysis load 10 uL of the resuspended beads and 1% of the lysate. To calculate 0.5% of the lysate, take the volume of lysate added multiply by 0.01, divide it by 5 (because you are only loading 1/5 of the resuspended beads) and multiply by 2 (if using 2X SDS cocktail on the lysate). For example if you loaded the beads with 500 uL lysate, you would run 2uL lysate. It may be necessary to dilute the lysate.
[[ Category: Protein-Protein Interactions ]]
[[ Category: Protein Biochemistry ]]
[[ Category: Proteins ]]
__NOTOC__
==Buffers==
Prepare each of these and chill on ice:
* Nucleotide Stripping Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 20 uL 0.5M EDTA
* Loading Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 100 uL 1M MgCl
* Lysis Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 4.5 mL water + 500 uL 20% Triton X100 + PI Tablet
* Wash Buffer: 25 mL [[ 2xHNG_Buffer | 2x HNG ]] + 25 mL water
==Preparation of Immobilized GTPases==
#Make 20 ug aliquots of GTPases immobilized to glutathione beads (see [[ GST Pulldown Assay ]])
#Resuspend one aliquot in 1 mL Nucleotide Stripping Buffer
#Incubate at Room Temperature for ~ 20min
#Spin down beads, aspirate supernatant and wash 1 x with 1 mL Loading Buffer.
#Aspirate supernatant and add 500 uL Loading Buffer plus 10 uL GDP or GTPyS
#Incubate at room temperature for about 20 min
==Preparation of Lysates==
#Wash cells 2x with 25 mL PBS (for 150 mm plate; use 10 mL for 100 mm dish)
#Add 2 mL Lysis buffer (or 1 mL for 100 mm dish) and scrape cells using plastic spatula. Collect in 1.5 mL eppendorf tubes
#Incubate end over end for 20 min at 4 C
#Centrifuge 10 min at 13 000 RPM at 4C to clarify
#Remove supernatants to fresh tubes.
#Save a lysate sample in 5X SDS-Cocktail
==Pull-Down Assay==
#Add appropriate amount of lysate to nucleotide-loaded beads. Typically we use 2 ug for a pull-down, so that corresponds to 1/5 of the resuspended GST-GTPase solution. You can vary the beads and the lysate but a good starting point is 500 ug protein lysate. If necessary bring volume of assay up to ~1 mL with HNG
#Add 50 uL of Glutathione Beads for bulk during washing.
#Incubate end over end at 4C for 30 min to 1h.
#Wash beads 5 times with 1 mL Wash Buffer
#Resuspend beads in 50 uL of 5X SDS Cocktail
#For analysis load 10 uL of the resuspended beads and 1% of the lysate. To calculate 0.5% of the lysate, take the volume of lysate added multiply by 0.01, divide it by 5 (because you are only loading 1/5 of the resuspended beads) and multiply by 2 (if using 2X SDS cocktail on the lysate). For example if you loaded the beads with 500 uL lysate, you would run 2uL lysate. It may be necessary to dilute the lysate.