...generate the phosphosites with serine instead of phosphoserine (i.e. a “phosphorylatable” phosphosite library... serine phosphorylation ( Rinehart Human Serine Phosphopeptide Library ). The phosphorylation sites (“...Reagents Rinehart Lab Phosphoprotein Reagents Protein phosphorylation is one of the most abundant forms of...questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code...or multiply phosphorylated proteins (Pirman et al., Nat Comm 2015). Improved phosphoprotein production ...(“phosphosites”) consist of a central phosphoserine site flanked by 15 amino acids on either side occurring... site-specific incorporation of phosphoserine in >36,000 phosphosites by tandem mass spectrometry (Barber...

...SDS-PAGE gels containing Phos-Tag, a small molecule that binds specifically to phosphate moieties and promotes... al. "Detection of phosphorylation on large proteins by Western blotting using Phos-tag containing gel...MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic dual...shift in phosphorylated, but not non-phosphorylated proteins (8). Quantifying the ratio of phospho- and non-phospho-bands...of the dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the...

...or dark conditions, the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription...histidine kinase/photosensor senses various light inputs, and an obligate chromophore allows the system...dark conditions, the chromophore activates the response regulator via phosphorylation, promoting transcription...in E. coli easier and more robust. Synthetic photobiology: why use light? In synthetic biology and bioengineering...TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses...two-component system (TCS). Levskaya et al. fused a photosensory domain from cyanobacteria to a common E. coli...hybrid construct senses light, and an obligate chromophore allows the system to respond to various light...

...dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and pa= photoactivation) domains on... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to the...-specifically activated and, like the game of telephone, often the signal must be transmitted by intermediate... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is...

...state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore and quencher...of aptamers means they can be used with or as fluorophores in several different ways. Researchers have ...fluorescent in situ hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. ...removing the quencher from the proximity of the fluorophore and subsequently leading to a positive signal...signal. The same effect can be achieved for a fluorophore that relies on dimerization for activation, only...used as powerful biosensors, the bound protein fluorophores and quenchers are still relatively bulky. One...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether...

...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...and efficiently photoconverted in plant, Drosophila, and mammalian cells. Photoconverted GFP is compatible...Davidson MW, Looger LL. A bright and photostable photoconvertible fluorescent protein. Nat Methods. 2009...accidental discovery that laser treatment can photoconvert GFP from green to RED! This simple technique...time-consuming to repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery...discovery that GFP itself is photoconvertible, new studies of spatiotemporal dynamics can be undertaken using...

...emitted photons before they are destroyed. Factors that affect photostability The photobleaching rate of...will discuss how to select a photostable fluorescent protein. Photobleaching is the irreversible destruction...destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some point...have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display...likelihood that a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does ... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...to compare the photostability of different fluorescent proteins? Measuring photostability Since it is clear...

...illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly to the anionic form, causing...presented first, it causes irreversible photoconversion of the chromophore from the neutral to the anionic form...mEosFP, can form it’s chromophore at a more comfortable 37°C (5). Dendra2 photoconverts from green to red ...reversible PA-FPs (also known as photoswitchers) can be photoswitched from a dark state to a bright fluorescent... for vanishing, and pa, for photoactivation) (6). Dronpa is photoactivated by blue wavelengths of light...consider, such as brightness, photostability, pH stability, chromophore maturation rate at 37 °C, turnover...-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce though...

...biophysical interaction between two fluorophores. When the first fluorophore (the donor) is excited, it can...acids/lipids of interest are fluorophore labeled and that these fluorophores can undergo FRET when they ...targeting a FRET fluorophore to a biomolecule is a bit like a Fedex delivery. Your fluorophore package needs... energy directly via resonance to the second fluorophore (the acceptor) if the two probes are close together...physical distance between the donor/acceptor fluorophores (Stryer, 1978), a measurement that is unaffected...label your biomolecule of interest with FRET fluorophores? When is absolute labeling specificity needed...measure it). Molecular mailbox: How to get your fluorophore delivered to the right place? FRET experiments...

...technique! What is a photosensitizer? First, for a few definitions. A photosensitizer is a dye or protein... higher photostability than KillerRed, with KillerRed reaching greater than 75% photobleaching after just...system? Turns out that with light-inducible photosensitizers and a quick zap of the proper color light,...light, you can do just that.  Photosensitizing dyes and proteins have been around for awhile (check out this...Bruchez and Tsang labs recently developed a photosensitizer composed of the protein complex complex and... oxygen species (ROS). The ROS produced by photosensitizers inactivate target proteins or cells while ...having little effect on neighboring cells. The photosensitizer used in this work is composed of two parts:...

...called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore can emit...the photon budget and pushing the frontiers of fluorescence microscopy often requires more photons. For...cells can decrease the number of fluorophores, compromising the photon budget. Likewise, the switch from...desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...more photostable than fluorescent proteins, providing a straightforward way to improve the photon budget...rhodamine fluorophores, such as the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and... of photons for your imaging experiment. Innovative labeling strategies and improved fluorophores are ...

...Listen to the photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which...., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop...ratio, high sensitivity, responds quickly, is photostable, and is compatible with optogenetic control. ... Discovery of new protein binder tools for photoswitchable proteins cPYP and AsLOV2 Article contributed...temporally control cellular processes in vivo. Many photoactive proteins exist in nature, but developing them... either the light- and dark- states of the photoswitchable cPYP, which changes in conformation in response...bind to only the light- or dark- state of the photoswitchable proteins offers a powerful approach to control...

...PLCG2 phospholipase C, gamma 2 (phosphatidylinositol-specific) - PPP3CA protein phosphatase 3 (formerly...IP1, IP2, IPD2, NEMO INPP5D inositol polyphosphate-5-phosphatase, 145kDa MGC104855, MGC142140, MGC142142...FLJ26625, JTK8 MALT1 mucosa associated lymphoid tissue lymphoma translocation gene 1 DKFZp434L132, MLT... PIK3CD phosphoinositide-3-kinase, catalytic, delta polypeptide p110D PIK3CG phosphoinositide-3-kinase...and cytoplasmic domain, (semaphorin) 6D FLJ11598, KIAA1479 SEMA7A semaphorin 7A, GPI membrane anchor (...BMP1 bone morphogenetic protein 1 FLJ44432, PCOLC, PCP, TLD, pCP-2 BMP10 bone morphogenetic protein 10...MGC126783 BMP15 bone morphogenetic protein 15 GDF9B, ODG2, POF4 BMP2 bone morphogenetic protein 2 BMP2A BMP3...

... Expansion Accessible Using Phosphoserine to Study Protein Phosphorylation Rinehart Lab reagents tRNA/... mutated, serB deleted for phosphoserine GCE. For expressing phosphoserine-containing proteins using genetic... pMAH2-CageCys leucyl-tRNA-synthetase E. coli photocaged cysteine Mammalian TAG Huiwang Ai 73544 pEvol-pAcFRS...91705 pSupAR-MbPylRS(DiZPK) PylRS M. barkeri photocrosslinkers DiZPK, DiZSeK, or DiZHSeC Bacterial TAG P....91706 pCMV-MbPylRS(DiZPK) PylRS M. barkeri photocrosslinkers DiZPK, DiZSeK, or DiZHSeC Mammalian TAG P.... PylT_FLAG-AbKRS-chIPYE chimeric PylRS Chimeric photo-crosslinking-lysine Mammalian TAG Simon Elsaesser...140022 pAS_4xMma PylT_FLAG-PcKRS PcKRS M. barkeri photocaged-lysine Mammalian TAG Simon Elsaesser 140023 pAS...

... curve with known amounts of protein. Fluorophores Fluorophores are light-sensitive molecules which emit...IF uses fluorophore color to differentiate between the targets of interest, if your fluorophore is bound...the fluorophores used is important. It is best to inversely pair the brightness of the fluorophore with...If a bright fluorophore is on an abundant target, it obscures fainter signals. Fluorophores can emit signals...storing your antibody: Antibodies conjugated to fluorophores should never be stored below 4 °C. Certain isotype...molecules are horseradish peroxidase (HRP) and fluorophores. Horseradish peroxidase (HRP) HRP performs a...specific wavelength. While more expensive than HRP, fluorophores are available in a range of colors activated...

...Webb Phosphate FRET-based phosphate sensor (FLIPPi) A novel analytical method for in vivo phosphate tracking...Ca(2+) indicator with enhanced two-photon absorption. Neurophotonics. 2024 Apr;11(2):024207. doi: 10.1117...29;7. pii: 32638. Kai Johnsson Phosphate Scaffold for inorganic phosphate biosensor Development of a Reagentless.... Eric Schreiter Calcium AAV expression of photoconvertible fluorescent protein-based calcium integrator...calcium sensor with exceptional brightness, photostability, and multiplexing capabilities. bioRxiv. 2025... jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths. Nat Methods...localized zinc sensor ZIBG2 Genetically Encoded, Photostable Indicators to Image Dynamic Zn(2+) Secretion ...

... Protein phosphatase 1 catalytic subunit alpha PREX2 Phosphatidylinositol-3,4,5-trisphosphate-dependent...receptor PDPK1 3-phosphoinositide dependent protein kinase 1 PEBP1 Phosphatidylethanolamine binding protein...PIK3R5 PIK3R6 Also known as PI3K; Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic and regulatory...state, regulated by the addition and removal of a phosphate group. Under normal physiological conditions GTPases...thymoma viral oncogene homolog 3 ALK Anaplastic lymphoma receptor tyrosine kinase ARHGAP35 Rho GTPase activating...DUSP2 DUSP3 DUSP4 DUSP5 DUSP6 Dual specificity phosphatase E2F E2F1 E2F2 E2F3 E2F transcription factor ECT2...G1, G2, G3: non-catalytic subunit gamma PTEN Phosphatase and tensin homolog RAC RAC1 RAC2 RAC3 Ras-related...

... Dlx EF1a/nEF Synapsin E2 regulatory element Fluorophore GFP Red-wavelength fluorescent protein Yellow-wavelength...pAAV-CaMKIIa-ChRmine-mScarlet-Kv2.1-WPRE CamKII ChRmine (high-photocurrent, red-shifted) mScarlet Constitutive 8 Deisseroth...pAAV-hSyn-ChRmine-mScarlet-Kv2.1-WPRE Syn ChRmine (high-photocurrent, red-shifted) mScarlet Constitutive 8 Deisseroth...pAAV-Ef1a-DIO-ChRmine-mScarlet-WPRE EF1a ChRmine (high-photocurrent, red-shifted) mScarlet Cre dependent 1, 5 Deisseroth... 6m-p2A-ChRmine-Kv2.1-WPRE Syn ChRmine (high-photocurrent, red-shifted) GCaMP6m Constitutive 8 Deisseroth... pAAV-nEF-ChRmine-mScarlet nEF ChRmine (high-photocurrent, red-shifted) mScarlet Constitutive 8 Deisseroth...pAAV-nEF-Con/Fon-ChRmine-oScarlet nEF ChRmine (high-photocurrent, red-shifted) oScarlet Cre and Flp dependent...

... multicolor imaging. Photo-activatable iRFPs can be ‘turned on’ by non-phototoxic far-red light and are...690/717 (after photoactivation) 3.2 pPAiRFP1-N1 PAiRFP2 692/719 (after photoactivation) 3.0 pPAiRFP2-N1... imaging using far red, near-infrared, and photoactivatable fluorescent proteins. Plasmid...imaging are derived from bacterial phytochrome photoreceptors (BphPs). Spectrally distinct permanently fluorescent...The following far red , near-infrared , and photoactivatable fluorescent empty backbone plasmids are ideal...piRFP720-N1 iSplit 690/713 5.3 pPAS-E and pK-GAFm Photoactivatable Protein Excitation/Emission Brightness Find...

... Large Stokes Shift , Photoactivatable , Photoconvertible , and Photoswitchable fluorescent proteins Various...monitor in live cells (beware of artifacts!), photoactivatable tags for super resolution imaging, epitope...20341 mEos2 (fluorescent protein) Loren Looger Photoactivable Fluorescent Protein Expression: contains mEos2...mEos2 with an N-terminal His tag. Photoactivable fluorescent proteins like this one can be used in super...protein, MS2. Can be used in combination with a fluorophore-tagged MS2 (such as that found in pZS*12-MS2-... to dsRED. Can be used in combination with a fluorophore-tagged MS2 (such as that found in pZS*12-MS2-...enhances the FRET signal from the CFP and YFP fluorophores linked by an arabinose binding protein (AraF...