...Metal spatula Razor blade Plastic tray Gel imaging system ImageJ or similar software Reagents Purified recombinant...the sink. Take a brightfield image of the gel with an appropriate imaging system. Figure 1 Recombinant... QC and would not be used. Section 3: Image Analysis Use ImageJ (Link opens in a new window) or a similar...bands to the overall lane. Import the gel image into ImageJ. Select File . Select Open . Choose the location...location of the file to open. Change the image type to 8-bit . Select Image . Select Type . Choose 8-bit . Determine... using ready-to-use biosafe Coomassie stain and ImageJ software. Protocols...250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing...

...expressed. Cells are then cultured and FACS sorted. Image from Mateo Sanchez and Alice Ting. Sanchez MI...sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. In their BioRxiv... adsorption chromatography (IMAC) using Ni-NTA magnetic beads. Workflow of nanobody expression and...genomic DNA with the help of the piggyBa transposase. Image from Hazelbaker et al., 2020. The lab tested ...wavelengths above 1,000 nm and enable two-color imaging with red fluorescence protein-based calcium sensors...

...photobleaching method and the fluorescence lifetime imaging microscopy (FLIM) method. Acceptor photobleaching...destroy the donor molecule. Fluorescence lifetime imaging microscopy method FLIM has been developed more ...acceptor. It should be noted that FLIM is a slower imaging method, limiting its use in many FRET experiments...interpreting data. FRET is 20 years old but as you can imagine not old-fashioned at all. With the development ...Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol...

...the fused XFP can be used to amplify the signal. Imaging endogenous fluorescence in live animals only permits...fluorophores and that can be combined with new imaging techniques.  References Cai, Dawen, et al. "Improved...17972876. Pan, Y. Albert, et al. "Multicolor Brainbow imaging in zebrafish." Cold Spring Harbor Protocols 2011.1...Addgene blog Read an introduction to live cell imaging Learn more about the origin of the Brainbow system...collection Learn about fluorescent tools for in vivo imaging ...

...trend in biology towards single-cell resolution imaging. Sanes and Lichtman endeavored to create a map ... these Brainbow constructs into mice. When they imaged the mouse brains, they found many more than 3-4...suffered from a number of limitations. First, the imaging of Brainbow mouse tissue was challenging due to...described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine Loulier, et ...given section of brain by at least an order of magnitude. It is clear that further refinements of the Brainbow...

...months at 4 °C.  Pro tip! If you get a speckled image, your protein-based blocker is either degraded or...to be compatible with stripping and probing.   Imaging At the final step of your western, you’ll have ...cost, but require no consumables, produce digital images, reach their limit of detection quickly (~1 minute...dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic range for fluorescent...Pro tip! You only need a resolution of ~150 µm to image a blot. This is because proteins diffuse semiradially...-based buffers Increased cost X-ray film imaging Increased time; increased consumables cost ...

... et al., 2008; Taylor et al., 2016; Hooper and Maguire, 2016). Recombinant adeno-associated viruses (rAAVs...1984; Van Duyne, 2001; Christenson Wick and Krook-Magnuson, 2018). Optogenetic FLEx vectors contain a strong... Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping." Journal of Neuroscience...Central PMCID: PMC4971574. Hooper, Andrew, and Jamie Maguire. "Characterization of a novel subtype of hippocampal...11340053. Wick, Zoé Christenson, and Esther Krook-Magnuson. "Specificity, versatility, and continual development...

...recovered from seven rescued F1 animals (gel image under worm). Image courtesy of Jordan Ward, UCSF. In ...2xFLAG knock-in by direct screening. The center images depict the power of lethal mutation co-selection...CRISPRa), visualize specific genomic loci (CRISPR-imaging), or to find the proteins associated with a given...could further transform the range of experiments imaginable in C. elegans. Equally, some of the “elegans”...

...microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I have worked...microbes by using non-invasive methods for cellular imaging with ultrasound. In his research he applies naturally...frequencies. Read more about ‘Using ultrasound to image bacteria in vivo: acoustic reporter genes’ on our...et al. "Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts." Nature 553.7686...

...injections are administered in the cisterna magna or lumbar spine. Image and caption courtesy of Challis et al...clover.caltech.edu. Figure adapted from Challis et al. 2022. Image created with BioRender.com. Abbreviations: AAV, ...This capsid was developed and deposited by the Maguire lab. In mice, it is sex-independent and does not...similar to humans than New World Monkeys and rodents. Image and caption adapted from Campos et al., 2023. ...Tsoulfas, P., & Chaudhari, N. (2022). Selectively Imaging Cranial Sensory Ganglion Neurons Using AAV-PHP....D., Dujardin, S., Ramirez, S. H., Hudry, E., & Maguire, C. A. (2019). Selection of an Efficient AAV Vector...

...world of fluorescent proteins and their use for imaging cell biology, Michael Davidson’s lab at Florida...been the go-to place. In 2012, his National High Magnetic Field Lab worked with an impressive 1,350 scientists...collection including some 3,300 plasmids along with image galleries and educational resources to go with them...Davidson lab even included some of their own reference images that can be found on the corresponding plasmid ...

...developed dLight1, a molecular tool to detect and image DA in live animals (Patriarchi et al., 2018). Similar...exhibit high (submicromolar) DA affinity and high-magnitude responses (increases in fluorescence) at low concentrations...et al., 2018). These sensors also exhibit high magnitudes of fluorescent responses. Studies on the kinetics...Patriarchi, Tommaso, et al. "Ultrafast neuronal imaging of dopamine dynamics with designed genetically ...

...penetrance and live cell imaging capabilities. Upon developing a new imaging-based bacterial colony screening...fluorescent proteins were the state of the art in RNA imaging in vivo. These new “Riboglow” probes contain a ...and achieve better signal in vivo as compared to imaging with broccoli aptamers or MS2 tags using the same...

...your application. Depending on the tag, directly imaging or tracking your protein may also be possible, ...peptide (YPYDVPDYA) derived from human influenza hemagglutinin. The tag has two negatively charged amino acids...fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry, or immunofluorescence where...western blot as monoclonal antibodies also exist for imaging. The obvious con of GFP is its size – which can...

...sterile conditions where disturbing the tissue for imaging or collection could contaminate it. I have used...self-cleaving 2A site linkages and a terminator. Image from He, Y., Zhang, T., Sun, H. et al. Hortic Res... highly expressing RUBY plant is a vibrant red/magenta while (bottom panel, left) a lower expressing RUBY... He has a passion for microscopy and beautiful imaging.   References and Resources References  He, Y....

...will produce a secondary color (yellow, cyan, or magenta) depending on the hairpin combination. A gRNA containing...the same sgRNA will generate additional colors. Image from Ma et al., 2016 with permission.   Developing...-arm (T1), and the q-arm (T2) of chromosome 19. Image from Ma et al., 2018 with permission.   Why...RNA scaffolds for signal amplification in genome imaging.” Nature Methods 15 (2018): 928-931. PubMed PMID...

... event. What are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital...that are released from cells during production (Maguire et al., 2012). They saw that these AAV particles...development of the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to...: 28082074. PubMed Central PMCID: PMC5368844.  Maguire, Casey A., et al. "Microvesicle-associated AAV ...

...and co-transfected into cells for reconstitution. Image from Levy et al., 2020 with permission. Though...quenches a proportion of mScarlet's emission via FRET. Image from Beck and Gong, 2019. Broadening the spectral...and subsequent transformation into cyanobacteria. Image from Vasudevan et al., 2018. Find the CyanoGate...binding, bringing the two halves of Cre together. Image from Eisenhoffer et al., 2019. This novel tool...

...membrane. The haemagglutinin (HA) tag is a small 9 amino acid epitope of the haemagglutinin of the influenza...membrane.   Figure 1: Electron microscope images showing ultrastructural localization of hM4Di in...coupled to mCherry (top) or HA tag (bottom). The images show examples of dendrites (blue shades) in the...

...   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy in HeLa...COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under CC-BY license...identification and characterization of AAV.CAP-Mac. Image reproduced from Chuapoco, Flytzanis, Goeden, et ...the most versatile. Whether you plan to purify or image a recombinant protein, the FLAG tag has proven itself...