Magnetic RFP-Trap®_M for Immunoprecipitation of ... - ChromoTek

Magnetic RFP-Trap ® _M for Immunoprecipitation of
RFP-Fusion Proteins
For the immunoprecipitation of RFP-fusion-proteins from cellular extracts.
Only for research applications, not for diagnostic or therapeutic use
1. Introduction
2. Content
Red fluorescent proteins (RFP) and variants thereof are widely used to study protein
localization and dynamics. For biochemical analyses including mass spectroscopy and
enzyme activity measurements these RFP fusion proteins and their interacting factors can be
isolated fast and efficiently (one step) via immunoprecipitation using the RFP-Trap ® . Since the
interaction is mediated by a small RFP binding protein coupled to magnetic particles the RFP-
Trap ® _M enables purification of any protein of interest fused to RFP (monomeric derivates of
DsRed, including mRFP1, mCherry, mPlum, mOrange).
RFP-Trap ® _M (magnetic particles, 3 mg/ml, size ~ 0.5 -1 µM in PBS, 0.05% NaAz),
Catalog no.: rtm-20; 0.5 ml; 20 reactions
binding capacity: 10 µl RFP-Trap ® _M binds 0.25 – 0.5 µg of RFP (data obtained from
HEK293T cells expressing RFP only).
3. Stability and
Storage
4. Protocol
Store material at 2-8°C, do not freeze, expiration time: 6 months after opening
1. For one immunoprecipitation reaction resuspend cell pellet (~10 7 cells) in 50 - 200 µl lysis
buffer by pipetting (or using a syringe)
2. Place the tube on ice for 30 min with extensively pipetting every 10 min
3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C
4. Transfer supernatant to a pre-cooled cup. Adjust volume with dilution buffer to 250 µl –
1000 µl. Discard pellet.
The cell lysate can be frozen at this point for long-term storage at -80°C.
For immunoblot analysis dilute 10 - 50 µl cell lysate with 50 µl 4x SDS-sample buffer
(-> refer as input)
5. Equilibrate RFP-Trap ® _M in dilution buffer (if necessary). Resuspend magnetic particles
and transfer calculated volume (20 - 30 µl) in a new reaction cup with 250 µl ice cold
dilution buffer . Magnetically separate until supernatant is clear and wash twice with 250
µl of cold dilution buffer
(note: magnetic separation can be improved by adding 0.01% detergent (f.c.) to the
dilution buffer)
6. Add cell lysate (200 – 1000 µl) to equilibrated RFP-Trap ® _M
7. Incubate the RFP-Trap ® _M with the cell lysate under constant mixing for 10 min – 2 h at
room temperature or 4°C
(note: during incubation of protein sample with the RFP-Trap ® _M the final concentration
of detergents should not exceed 0.2% to avoid unspecific binding to the matrix).
8. Magnetically separate until supernatant is clear
9. For western blot analysis dilute 50 µl supernatant with 50 µl 4x SDS-sample buffer
(-> refer as non-bound). Discard remaining supernatant
10. Wash magnetic particles two times with 250 µl – 400 µl ice cold wash buffer
(optional: increase salt concentration in the second washing step up to 500 mM)
(note: magnetic separation can be improved by adding 0.01% detergent (f.c.) to the wash
buffer )
RFP-Trap ® _M manual - 1 - Version 2011-05-10

11. Resuspend magnetic particle in 100 µl 2x SDS-Sample buffer
12. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from
the beads. Magnetically separate the RFP-Trap ® _M and transfer supernatant to a fresh
cup. SDS-PAGE should be performed with the supernatant.(-> refer as bound)
13. (optional) elute bound proteins by adding 50 µl 0.2 M glycine pH 2.5 (incubation time: 30
sec under constant mixing) followed by magnetic separation. Transfer the supernatant to
a fresh cup and add 5 µl 1M Tris-base (pH 10.4) for neutralization. To increase the elution
efficiency this step can be repeated.
Suggested Buffers (as tested in our laboratory)
Lysis-buffer (for CoIP):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
0.5% NP40
1 mM PMSF freshly added (optinal)
1x mammalian Protease Inhibitor Cocktail (e.g. Serva ® ) freshly added
(optional for nuclear proteins / chromatin proteins:
DNaseI final conc. 1 µg/µl
2.5 mM MgCl 2 )
Dilution-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (options)
1x Protease Inhibitor Cocktail (e.g. Serva) freshly added
Wash-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.01% NP-40
0.5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva ® ) freshly added
RIPA-Buffer (for cell lysis):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.1% SDS
1% TX100
1% Deoxycholate
5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva ® ) freshly added
RFP-Trap ® _M manual - 2 - Version 2011-05-10