ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- |GST-Lysis Buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment) |- |GST-Pulldown/wash buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment) |- | |Purified recombinant..."

2025-02-27T12:16:55Z

Created page with "{| class="wikitable" |+ !Materials ! |- |GST-Lysis Buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment) |- |GST-Pulldown/wash buffer: |50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment) |- | |Purified recombinant..."

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{| class="wikitable"
|+
!Materials
!
|-
|GST-Lysis Buffer:
|50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add protease and phosphatase inhibitors freshly on the day of the experiment)
|-
|GST-Pulldown/wash buffer:
|50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 0.05% sodium deoxycholate, 1% Triton- X, 10 µM ZnCl2, 50mM NaF (add 1mM DTT freshly on the day of the experiment)
|-
|
|Purified recombinant proteins
|-
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|Glutathione sepharose resin
|}

=== Procedure ===
----

==== Equlibration of beads ====
Glutathione beads are supplied in 20% ethanol. Before use, the resin must be washed and prepared as a slurry consisting of one part beads and two parts GST- Pulldown Buffer as described in step 1-4. Larger amounts can be prepared and stored at 4°C.

* Gently mix the resin by inverting the flask until you obtain a homogenous solution. Use 10 µl of resin per reaction and transfer into a fresh Eppendorf tube
* Collect the beads by centrifugation at 2700 g at 4°C for 2 min (faster and longer spins can fragment the beads). Remove the supernatant by aspirating carefully using a glass Pasteur pipette attached to vacuum line.
* Wash the beads 3 times with 500 µl of GST-Pulldown Buffer. To perform each wash, add Pulldown Buffer, mix gently, centrifuge at 2700 g for 2 min and aspirate the supernatants
* After the last wash add two bead volumes of GST-Pulldown Buffer resulting in a 50% bead slurry and store on ice

==== Precleaning of lysate ====

* Lyse cells according to whole cell lysate protocol with the following changes:
** Use GST-Lysis Buffer for cell lysis
** After mechanically shearing the DNA add 20 µl Sepharose beads + 20 µl glutathione sepharose beads + 25 µg of GST to cell lysate and incubate for up to 30 min at 4° on head-to-head rotor.
** Centrifuge at 13.000 rpm at 4° for 5 min
** Distribute equal amounts of supernatant into fresh Eppendorf tubes. (Two tubes/sample are required at minimum: one will be incubated with the GST-bait protein and a second with control GST protein.)

==== Pulldown ====

* Centrifuge the protein extracts at 10.000 g at 4 °C for 20 min. Transfer supernatants to fresh microcentrifuge tubes on ice (This step is necessary to remove precipitated protein which can happen due to multiple freeze thaw cycles).
* Add 20 µl the 50% bead slurry from step 4 and 2-5 µg of GST-protein to the samples. '''Equimolar ratios of bait and control protein should be used!!'''
* Incubate for 2h at 4° on head to head rotor

GST-Pulldown - Revision history