TY - CHAP
T1 - Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells
AU - Van Royen, Martin E.
AU - Farla, Pascal
AU - Mattern, Karin A.
AU - Geverts, Bart
AU - Trapman, Jan
AU - Houtsmuller, Adriaan B.
PY - 2008
Y1 - 2008
N2 - Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.
AB - Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.
UR - https://www.scopus.com/pages/publications/60349125976
U2 - 10.1007/978-1-60327-461-6_20
DO - 10.1007/978-1-60327-461-6_20
M3 - Chapter
AN - SCOPUS:60349125976
SN - 9781603274609
SN - 978-1-61737-898-0
VL - 2
T3 - Methods in Molecular Biology
SP - 363
EP - 385
BT - The Nucleus
PB - Humana Press
ER -