Larry Goldfinger
Protocol
Project: Ras activation
Title: Ras activation by GST-RBD pulldown.
Purpose: to assay temporal kinetics of H-Ras activation in cells.
Approach: Use GST-RBD (Ras-binding domain of Raf-1) in a pulldown assay with GSH beads to extract
GTP-H-Ras from cell lysates.
Materials and Reagents:
Name
-H-Ras, rb
RLB (see Buffers)
PI cocktail
GTP
Raf-RBD
GSH sepharose
5x/1.5X SDSB
4-20% gel
DAM-IR 680
Blotto
Date
Vendor
Cat. #
SCBT
C-20
Roche
Inv.
LICOR
LICOR
Lot. #
Conc.
Thawed
/Psg #
Made by
200 g/mL
20x
10 mM
500ng/L
Fresh
EC60255BOX
926-32213
1x
Buffers:
Ras lysis buffer (RLB): 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Na DOC, 10% glycerol, 2 mM
EGTA, 2 mM MgCl2.
Procedure:
Prepare complete lysis buffer RLB by adding fresh PI cocktail, 10 M GTP, and freshly thawed GST-RafRBD protein (0.5 g/L).
You will need 500 L complete RLB per 100 mm dish of confluent cells. Add enough GST-RBD
protein to give 20 g per sample. 
Stimulate cells according to the experiment.
Place immediately on ice.
Immediately add 500 L ice cold RLB + freshly added PI cocktail, GTP and GST-RBD.
Incubate on ice 10 minutes.
Centrifuge at full speed for 5 min at 4°C.
Transfer supernatant to new tube. Discard pellet.
Save 20 L for WCL sample. GST-RBD pulldown with remaining lysates.
Ras pulldown Procedure:
Always cut off the end of the pipet tip with a razor blade at an oblique angle before pipetting GSHsepharose.
Pre-equilibrate GSH-sepharose in RLB by washing several times and spinning down. Resuspend in RLB to
give a 1:1 beads:buffer suspension. Mix thoroughly before using.
1. Add 40 L GSH-sepharose 1:1 suspension to each sample.
2. On rocker at 4°C, 2 hrs.
2/7/2016
Refer
3.
4.
5.
6.
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Wash all bead fractions with 400 L RLB + 10 M GTP, WITHOUT GST-RBD, five times.
Aspirate last wash.
Resuspend beads in 50 L 1.5x SDS-PAGE buffer + DTT.
Boil samples and SDS-PAGE WCL and pulldown samples, like the example below.
Gel 1
Sample
MWM
WCL 1
WCL 2
WCL 3
WCL 4
WCL 5
WCL 6
WCL 7
WCL 8
WCL 9
WCL 10
WCL 11
MWM
MWM
Amount
1
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
1
1
Gel 2
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Sample
MWM
RBD 1
RBD 2
RBD 3
RBD 4
RBD 5
RBD 6
RBD 7
RBD 8
RBD 9
RBD 10
RBD 11
MWM
MWM
Amount
1
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
15 L
1
1
Blot:
1° Ab: rb anti-HRas 1:1000 each, 2 hrs.
2° Ab: DAR800 1:10000 each, 1 hr.
Expected Results: HRas = 21 kDa but usually runs closer to 18 kDa. H-Ras should be present in Raf-RBD
pulldowns from the stimulated cells – presence of H-Ras in these samples indicates GTP-Ras (i.e.,
activated Ras).
Results:
Conclusions: 
Future directions, experiments, problems:
2/7/2016