4. Cut off the end of the tips when pipetting beads.
5. The amount of beads can be adjusted to the amount of
RBP-GFP fusion in the cellular extract. We obtain similar
results by using GFP-Trap
® coupled to magnetic beads. A
systematic investigation of proteins binding nonspecifically to
commonly used affinity matrices, quite appropriately termed
the “bead proteome,” showed that less unspecific binding for
cytoplasmic extracts occurred on Sepharose or agarose beads
whereas magnetic beads showed lower nonspecific binding for
nucleic acid-associated proteins and, thus, nuclear extracts [8].
6. Preparation of beads (see Subheading 3.3) has to be completed
before the extracts are prepared.
7. Use DNALo Bind Tubes (Eppendorf) to avoid unspecific binding of proteins and nucleic acids to the walls of the tube during
IP. Change tubes during IP to remove residual proteins and
nucleic acids which bind unspecifically to the walls of the tube.
8. Washing conditions have to be established carefully to reduce
unspecific binding as much as possible but to avoid dissociation
of the specific RNA-protein interactions and of the
RBP-antibody interaction. Stringency of washing buffer can
be changed by adjusting the concentration of NaCl, urea, or
other chaotropic salts, e.g., LiCl.
9. Recovery of RNA is most efficient by using Tri-Reagent. The
use of commercial kits based on column purification of RNA is
not recommended.
10. We incubate the samples at 55
C to substitute for the heat
treatment commonly used to reverse the formaldehyde crosslinking [9].
11. Be careful when washing the RNA pellet and decanting the
supernatant because the pellet can detach from the wall of the
tube very easily.
12. Depending on the amount of coprecipitated RNA, we combine the RNA of up to six individual RIPs per biological
replicate.
Acknowledgements
This work was supported by the DFG (STA 653/6 and STA653/9).
460
Tino Ko ¨ ster and Dorothee Staiger
5. The amount of beads can be adjusted to the amount of
RBP-GFP fusion in the cellular extract. We obtain similar
results by using GFP-Trap
® coupled to magnetic beads. A
systematic investigation of proteins binding nonspecifically to
commonly used affinity matrices, quite appropriately termed
the “bead proteome,” showed that less unspecific binding for
cytoplasmic extracts occurred on Sepharose or agarose beads
whereas magnetic beads showed lower nonspecific binding for
nucleic acid-associated proteins and, thus, nuclear extracts [8].
6. Preparation of beads (see Subheading 3.3) has to be completed
before the extracts are prepared.
7. Use DNALo Bind Tubes (Eppendorf) to avoid unspecific binding of proteins and nucleic acids to the walls of the tube during
IP. Change tubes during IP to remove residual proteins and
nucleic acids which bind unspecifically to the walls of the tube.
8. Washing conditions have to be established carefully to reduce
unspecific binding as much as possible but to avoid dissociation
of the specific RNA-protein interactions and of the
RBP-antibody interaction. Stringency of washing buffer can
be changed by adjusting the concentration of NaCl, urea, or
other chaotropic salts, e.g., LiCl.
9. Recovery of RNA is most efficient by using Tri-Reagent. The
use of commercial kits based on column purification of RNA is
not recommended.
10. We incubate the samples at 55
C to substitute for the heat
treatment commonly used to reverse the formaldehyde crosslinking [9].
11. Be careful when washing the RNA pellet and decanting the
supernatant because the pellet can detach from the wall of the
tube very easily.
12. Depending on the amount of coprecipitated RNA, we combine the RNA of up to six individual RIPs per biological
replicate.
Acknowledgements
This work was supported by the DFG (STA 653/6 and STA653/9).
460
Tino Ko ¨ ster and Dorothee Staiger