Conditions for using FRAP as a quantitative technique--influence of the bleaching protocol

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Dominika O TrembeckaJurek W Dobrucki

Abstract

Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent way of data analysis.

References

Mar 8, 1991·Science·C C CunninghamD J Kwiatkowski
Dec 29, 2000·Nature·T MisteliD T Brown
Dec 18, 2004·Methods in Cell Biology·Jurek W Dobrucki
Apr 26, 2005·Biophysical Journal·Valérie M LaurentJean-Jacques Meister
Sep 6, 2007·The Journal of Biological Chemistry·Allen KaasikVladimir Veksler
Dec 25, 2007·Methods in Cell Biology·James G McNally
Feb 13, 2008·IEEE Transactions on Image Processing : a Publication of the IEEE Signal Processing Society·P ThévenazM Unser
Sep 6, 2008·Molecular and Cellular Endocrinology·Joanna Dutko-GwóźdźAndrzej Ozyhar
Mar 25, 2009·The Journal of Physical Chemistry. B·Katrin G HeinzePaul W Wiseman
Jul 1, 2009·The Journal of Cell Biology·Jennifer C Waters
Jul 10, 2009·Cell Cycle·Nicole HappelDetlef Doenecke

❮ Previous
Next ❯

Citations

Mar 29, 2013·Nucleic Acids Research·Katrin SchneiderLothar Schermelleh
Feb 14, 2013·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Ewa BielaJurek W Dobrucki
Nov 4, 2010·Biophysical Journal·Timothy J StasevichJames G McNally
Sep 1, 2015·Quarterly Reviews of Biophysics·Niklas LorénKevin Braeckmans
Jan 23, 2013·Nucleus·Dominika O Trembecka-LucasJurek W Dobrucki
Oct 25, 2012·Current Protocols in Neuroscience·Lori R Hardy

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.