ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! !''add fresh'' |- |Lysis Buffer |Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10% | rowspan="2" |DTT 1 mM, Leupeptin, Aprotinin, PMSF |- |Wash Buffer |Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl₂ 30 mM, NP40 1% |- | |Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD) | |} === Procedure === ---- * Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice. * Stimu..."

2025-02-27T12:27:51Z

Created page with "{| class="wikitable" |+ !Materials ! !''add fresh'' |- |Lysis Buffer |Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10% | rowspan="2" |DTT 1 mM, Leupeptin, Aprotinin, PMSF |- |Wash Buffer |Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl<sub>2</sub> 30 mM, NP40 1% |- | |Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD) | |} === Procedure === ---- * Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice. * Stimu..."

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{| class="wikitable"
|+
!Materials
!
!''add fresh''
|-
|Lysis Buffer
|Tris pH 7.5, 50 mM, NaCl 150 mM, MgCl2 10 mM, NP40 1%, Glycerol 10%
| rowspan="2" |DTT 1 mM, Leupeptin, Aprotinin, PMSF
|-
|Wash Buffer
|Tris pH 7.5, 25 mM, NaCl 40 mM, MgCl<sub>2</sub> 30 mM, NP40 1%
|-
|
|Glutathione-Sepharose beads with bound recombinant GST-PAK-binding-domain (GST-PBD)
|
|}

=== Procedure ===
----

* Prepare lysis and wash buffer and keep on ice. Thaw beads (-80°C) on ice.
* Stimulate cells to activate Rac (e.g. phagocytosis, cell spreading) For small cells (e.g. HL60) use 1-2 x 107 cells/pulldown, for larger cells less
* Lyse cells on ice in 600 ul complete lysisbuffer and incubate 20 min on ice
* Centrifuge lysates for 10 min at 14k rpm at 4°C
* Transfer 500 ul supernatant to new pre-chilled tube on ice
* Keep the remaining supernatant as “input” samples (60 ul sup + 20 ul 4x SDS- LB)
* Add 20 ul PBD-beads (use cut-off yellow tip) to each 500 ul supernatant
* Incubate pulldowns for 45-60 min on rotating wheel in the cold room (4°C)
* Centrifuge the lysates for 2 min at 2000 x g
* Remove the supernatant carefully (don’t disturb the pelleted beads)
* Wash each bead pellet 3 times with 500 ul ice-cold washing buffer
* Resuspend the beads in 30 ul 2x SDS-LB and boil in a shaking heat block (7 min at 99°C, 1000rpm)
* Centrifuge the beads after boiling for 5 min at 14k rpm
* Transfer the supernatant to a new tube (try NOT to transfer any beads)
* Run samples on a 12.5% SDS-PAGE gel with large slots. Load the complete pulldown sample (30-40 ul) and 30 ul of the input samples.
* Transfer to PVDF (Western Blot) and use an anti-Rac antibody to detect GTP- Rac (pulldown samples) and total Rac (input samples)

Rac pulldown assay (with GST-PBD beads) - Revision history