GST pull down
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References: Arnaud Mouchon, Chris Depoix, Bruno Lefebvre
Purpose and comments: Assay for protein:protein interaction in-vitro
Bl21 are generally difficult to transform, at least as purchased from Promega and sometimes fromNovagen. Do not hesitate to use 2-3 _ g DNA for 50 _ l bugs. You must have in those conditions a platewith many colonies. If not, carry out another transformation but do not try to overexpress your protein fromlow efficiency transformed bugs, it won’t simply work. When comparing different receptors in your pulldown, do not omit to check how much protein is synthesized by TCA precipitation
Use fresh IPAB buffer
Always add DTT fresh to the IPAB and PBS buffer.
The trick: if you want to study ligand-dependent dimerization of RAR : RXR without DNA, your wash buffer must be 1x PBS supplemented with 500mm NaCl.
Material and solutions:
IPAB-gelatin: HEPES 20mM pH7.8
KCl 150mM
Gelatin 0.1%
TritonX100 0.1%
NP40 0.1%
MgCl2 5mM
DTT 2mM
Wash buffer:
PBS 1X
DTT 2mM
TnT Quick T7 or SP6 Kit from PROMEGA
Glutathion-Agarose beads from Pharmacia
Protocol:
a) Resin preparation (50% slurry):
1- Transfer a 1.33 mL aliquot of the resin (mix well before use) into a 15 mL Falcon tube
2- Centrifuge (1pulse at 500g)
3- Resuspend in 10 mL PBS 1X
4- Centrifuge (1pulse at 500g)
5- Resuspend in 10 mL PBS 1X (stable for one month at 4 _ C)
b) Extract preparation:
1- Transform BL21 or any appropriate bacterial strain with your GST-fused cDNA. Always use
freshly transformed cells
2- Grow a 50mL culture overnight
3- Seed a 1 to 2 liters culture the next morning
4- At OD600 _ 0.8, induce with 1mM IPTG for three hours
5- Pellet bacteria (20 min at 4000rpm, 4 _ C)
6- Freeze down the bacterial pellet for at least 30 min
7- Resuspend the bacterial pellet in 10 mL/liter of culture and vortex. BL21 should undergo lysis
very easily, other bacterial strain might require the use of lysozyme and/or DOC up to 0.5%.
8- Spin down cell debris for 30 min at 25,000x g
9- Collect the sup and bring to 10% glycerol
10- Store in 1 mL aliquots at -80 _ C
c) Affinity matrix preparation
1- Add 100_ L slurry for 1 mL extract
2- Agitate for 30 min on a spinning wheel at RT
3- Wash three times with 1x PBS
4- Resuspend the resin in 100 _ L IPAB-gelatin bufferl
d) GST- pull down
1- In an Eppendorf add:
5_ L of in-vitro synthesized receptor ( 35 S-labelled)
Usual conditions for RAR & RXR as well as for GR are as follows:
-TnT Quick Master Mix: 10 _ L
-DNA 1_ g/_ L: 0,8 _ L
- 35 S Méthionine: 0,5 _ L
-Nuclease Free Water: 1,2 _ L
Incubate for 60 min at 30 _ C
2- Bring to 150_ L with IPAB-Gelatin buffer supplemented with 2mM DTT
3- Add 1 _ M ligand when required
4- Incubate 30min at 4 _ C in the dark if using light-sensitive compounds
5- If required, bring to 1pM RARE or any HRE you need
6- Incubate 30min at 4 _ C
7- Add 50_ L of the GST affinity matrix
8- Incubate for 2 hours at 4 _ C
9- Wash twice with PBS-DTT buffer (quick pulse to 500 x g)
10- Add 25_ L of SDS loading buffer directly onto the pelleted resin
11- Vortex
12- Heat for 1min at 100 _ C
13- Analyze by SDS-PAGE and autoradiography
Purpose and comments: Assay for protein:protein interaction in-vitro
Bl21 are generally difficult to transform, at least as purchased from Promega and sometimes fromNovagen. Do not hesitate to use 2-3 _ g DNA for 50 _ l bugs. You must have in those conditions a platewith many colonies. If not, carry out another transformation but do not try to overexpress your protein fromlow efficiency transformed bugs, it won’t simply work. When comparing different receptors in your pulldown, do not omit to check how much protein is synthesized by TCA precipitation
Use fresh IPAB buffer
Always add DTT fresh to the IPAB and PBS buffer.
The trick: if you want to study ligand-dependent dimerization of RAR : RXR without DNA, your wash buffer must be 1x PBS supplemented with 500mm NaCl.
Material and solutions:
IPAB-gelatin: HEPES 20mM pH7.8
KCl 150mM
Gelatin 0.1%
TritonX100 0.1%
NP40 0.1%
MgCl2 5mM
DTT 2mM
Wash buffer:
PBS 1X
DTT 2mM
TnT Quick T7 or SP6 Kit from PROMEGA
Glutathion-Agarose beads from Pharmacia
Protocol:
a) Resin preparation (50% slurry):
1- Transfer a 1.33 mL aliquot of the resin (mix well before use) into a 15 mL Falcon tube
2- Centrifuge (1pulse at 500g)
3- Resuspend in 10 mL PBS 1X
4- Centrifuge (1pulse at 500g)
5- Resuspend in 10 mL PBS 1X (stable for one month at 4 _ C)
b) Extract preparation:
1- Transform BL21 or any appropriate bacterial strain with your GST-fused cDNA. Always use
freshly transformed cells
2- Grow a 50mL culture overnight
3- Seed a 1 to 2 liters culture the next morning
4- At OD600 _ 0.8, induce with 1mM IPTG for three hours
5- Pellet bacteria (20 min at 4000rpm, 4 _ C)
6- Freeze down the bacterial pellet for at least 30 min
7- Resuspend the bacterial pellet in 10 mL/liter of culture and vortex. BL21 should undergo lysis
very easily, other bacterial strain might require the use of lysozyme and/or DOC up to 0.5%.
8- Spin down cell debris for 30 min at 25,000x g
9- Collect the sup and bring to 10% glycerol
10- Store in 1 mL aliquots at -80 _ C
c) Affinity matrix preparation
1- Add 100_ L slurry for 1 mL extract
2- Agitate for 30 min on a spinning wheel at RT
3- Wash three times with 1x PBS
4- Resuspend the resin in 100 _ L IPAB-gelatin bufferl
d) GST- pull down
1- In an Eppendorf add:
5_ L of in-vitro synthesized receptor ( 35 S-labelled)
Usual conditions for RAR & RXR as well as for GR are as follows:
-TnT Quick Master Mix: 10 _ L
-DNA 1_ g/_ L: 0,8 _ L
- 35 S Méthionine: 0,5 _ L
-Nuclease Free Water: 1,2 _ L
Incubate for 60 min at 30 _ C
2- Bring to 150_ L with IPAB-Gelatin buffer supplemented with 2mM DTT
3- Add 1 _ M ligand when required
4- Incubate 30min at 4 _ C in the dark if using light-sensitive compounds
5- If required, bring to 1pM RARE or any HRE you need
6- Incubate 30min at 4 _ C
7- Add 50_ L of the GST affinity matrix
8- Incubate for 2 hours at 4 _ C
9- Wash twice with PBS-DTT buffer (quick pulse to 500 x g)
10- Add 25_ L of SDS loading buffer directly onto the pelleted resin
11- Vortex
12- Heat for 1min at 100 _ C
13- Analyze by SDS-PAGE and autoradiography
