...cross-linking cellular proteins. Cross-linking is a chemical reaction that covalently joins two molecules and... polymerize and lose its ability to cross-link proteins. As such, you will get the best results if you...solvents can be quite harsh - lipids and soluble proteins can be lost during fixation with these chemicals...these chemicals can also alter the structure of proteins, which could disrupt your target epitope (Fig....Formaldehyde cross-links the protein (red lines), which maintains structural epitopes (orange region), but it can...Permeabilization What is the point?  Antibodies are large proteins that need a little help crossing cell membranes...binding to any target protein that is still on the inside of the cell. However, assuming you do need to permeabilize...

...reasons arguing against this approach. First, this would prevent the study of the proteins independently...viral proteins (compared to viral infection). Therefore, the roles/functions of individual proteins can ...sense RNA strand approximately 19 kb in length. It contains short promoter sequences at its 3’ and 5’ ends... viral gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription...is a shortened version of the viral genome. It contains the 3’ and 5’ ends of the genome which are required...start and gene end sequences. Thus, the minigenome contains all cis-acting signals necessary to direct minigenome...transcribed in the cell when the appropriate viral proteins are provided in trans (see Other Important Components...

...month to a year. The researcher remains employed by their home institute, meaning the host university does... host lab or institute provides something that you can’t obtain at your current institute. A common reason...scientist, in academia, is someone who visits an institution to perform research there temporarily. The length...requires re-location of the scientist to the new institute where their typical workdays will be carried out...back to your home lab. Alternatively, the host institute may have a piece of equipment that is custom-built...train and acquire the data you need on the new instrumentation. Bonus reasons Visiting gives you access to...level researchers, it can expose them to a new institute and mentor that they may want to consider for ...

...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs...Kroupova et al., 2024). This construct contains the Lon and TBD domains, part of the HB domain, and several... tag antibody produced by our partners at the Institute for Protein Innovation (IPI), part of a new collection...short, linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987)...a fusion of mouse variable and rabbit constant domains and is recommended for use in western blots (Figure... own antibody or want to swap out the constant domains, the plasmids for the heavy chain and light chain...Antibody here!   Anderson, Z., Zhu, H., Riedel, T., Moshinsky, D., Meijers, R. (2024). Western Blot for Anti-V5...

...coding regions. In total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive...in different mouse strains. The in vivo selection for PHP.B in C57BL/6J mouse strains likely contributed...transduction of AAV serotypes generally tracks between strains of mice. This suggests that a small genetic difference...phenotypes. Two labs, the Deverman lab at the Broad Institute and the Wilson lab at the University of Pennsylvania...mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library was then...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV ...searched for genetic difference between the 36 mouse strains that are part of the Mouse Genome Project. They...

...be limited by a few caveats. Since fluorescent proteins (FP) require external light activation, you can...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene...including optogenetics, biosensors, and fusion proteins. Solving the problems of low luminescence By...generates much less signal than comparable fluorescent proteins. This weak emission hasn’t stopped some scientists...scientists from using chemiluminescent proteins for imaging, but it has precluded widespread adoption of the...eliminates the need for an excitation light source. Instead, a luciferase substrate like coelenterazine is ...Nano-lanterns and showed that these three luminescent proteins can be used together in cells. As Nano-lanterns...

...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk...access into the cell. The viral envelope and its proteins determine what type of cell the virus will target...can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo – ...Image courtesy of National Human Genome Research Institute. Its genome, however, isn’t enough to ensure...ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system to ...signals which can traffic their viral particles inside the nucleus, without the need for envelope disassembly...kb, but viral packaging is less efficient when inserts are on the higher end. Adeno and adeno-associated...

... antibody on crosslinked proteins. In the case of histone modifying proteins, which bind quite strongly...that ChIP uses native proteins (so antibodies validated only for denatured proteins may not be a good choice...another. For instance, a sample may need more or less cross-linking to firmly attach the proteins to the DNA...is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works by using...exploring these interactions, and can give valuable insight into DNA expression and protein interactions, including...the downstream analysis process. And ChIP itself begins at...  Crosslinking Once you have your sample, ...other until you are ready to separate them. Most proteins are not likely to remain attached to the DNA throughout...

...Takeda et al., 2002). G proteins: The receptor is coupled with heterotrimeric G proteins on the intracellular... catalyzes the biotinylation of proteins near the GPCR. These proteins are then identified with mass spec... into two parts: the receptor itself and the G-proteins it associates with.  Receptor: GPCRs are also...recognizes the appropriate ligand to activate the G proteins. It is estimated that there are approximately ...intracellular side of the cell membrane. Heterotrimeric G proteins are important molecular switches in signal transduction...subunits: alpha (Gα), beta (Gβ), and gamma (Gγ). G proteins are divided into four subtypes, based on their...role of 14 of the 16 non-visual heterotrimeric G proteins that can be activated downstream of GPCRs. This...

...joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often...repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs...much lower efficiency than NHEJ. ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Base ...the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Nick ...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type...homology-directed repair (HDR). ID Plasmid Description Gene/Insert Promoter Selectable Marker PI Publication Activate...activator to your specific locus. ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Interfere...

...S-shaped curve. Sparse proteins may not be detected at all, while overloaded proteins may saturate the detector...choose. Housekeeping proteins as loading controls Ubiquitously expressed proteins like β-actin or GAPDH... be careful relying on so called “housekeeping proteins.” Fully quantifying a western blot requires a ...semi-quantitative western blots. No pipetter is perfect, and proteins may not transfer precisely evenly across the entire...transfer errors — not to any factors from before the proteins even hit the gel. But what if your experiment ... this S-shaped relationship poses problems for proteins of very different abundance, consider the following...controls. These are often called “housekeeping proteins” and are detected with an antibodies, just like...

...why at Experimental Designs Consulting (EDC), we instead have our clients focus on their experimental needs...parties are clear on the goals and the resources. Instead of creating a start up budget, create a start up... a start up award by giving you step by step instructions on creating a budget, making decisions based...specific aims. This is actually why a department/institute is interested in you. Your science will define...greater than $5,000 but this can depend on the institution/funding source. Major equipment includes large...academic job titles in the regions where the host institute is located. For example, salaries in Kansas versus...is important to ensure that you’re looking at institutes that have the means to support your work. For...

...cells. The Brainbow-1.0 construct contains three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP...injection to insert these Brainbow constructs into mice. When they imaged the mouse brains, they found ...Brainbow-1.1 scales up the system to four fluorescent proteins, with recombination events specified by three ...colors (n-GFP, RFP, YFP or M-CFP.) The construct contains two tandem invertible segments, each with two ...The remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system,...intensity, caused partially by fluorophore photoinstability, as well as the tendency of fluorophores to... First, they screened a variety of fluorescent proteins to find those with ideal characteristics (low ...

...Plasmid Collections Fluorescent Proteins Organelle Markers Fluorescent Proteins: Subcellular Localization More...as these well-characterized proteins. General Structures The cell contains a variety of organelles and...role in secreting proteins from the cell to the extracellular environment. Proteins are first translated...Plasmids encoding fluorescent proteins tagged with genes or peptides with known subcellular localization...Fluorescent Protein Resources: Empty Backbones Allen Institute for Cell Science Plasmid Collection FRET Biosensors...has assembled a collection of fluorescent fusion proteins that are targeted to various organelles or subcellular...FAP; mCerulean3 Marcel Bruchez 45944 pTDpelB-C_sfYFPTwinStrep Periplasmic space PelB signal sequence YFP...

...Genetics 2014 Allen Institute for Cell Science Plasmid Collection The Allen Institute for Cell Science has...CRISPR system has been adapted to allow tagging of proteins expressed from their natural chromosomal context...add a tag (currently using FLAG) to endogenous proteins. This CRISPR/Cas plasmid-system consists of two...2A-PuroR cassette to the C-terminus of endogenous proteins. The PITCh system depends on MMEJ (microhomology-mediated...end-joining), an alternative DSB repair pathway, instead of HDR, which makes it easy to construct a donor...'s lab for building CRISPR targeting vectors to insert genetic tags in the C. elegans genome. The SapTrap...template for an individual tagging event. The kit contains 26 plasmids; 21 of the plasmids are for use in...

...the gene/insert by aligning to NCBI entry or using BLAST.   Confirming tags and fusion proteins. We’ll...of the plasmid.   Confirming the insert We usually confirm the insert through BLAST or by direct alignment...Confirming tags and fusion proteins Finally, we confirm promoters, tags, fusion proteins, and selectable markers...and user friendly, to own the process entirely remains a large undertaking. Priority number one was to...by the depositor. Depositing labs often provide insert sequences or annotated Genbank files that are useful...We look for point mutations, truncations, and insertions that could compromise function. When we do find...promoter, and rabbit beta-globin splice acceptor, contains sequence that is over 80% GC. Some IRES sequences...

...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers...optogenetics.  Common opsins used in zebrafish Optogenetics functions through opsins which are light sensitive...sensitive proteins. Opsins used in optogenetic studies are not native to vertebrates and are commonly introduced...fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks of life...system for behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...

... primers used to amplify the insert have sequences specific to the insert with an additional 15-17 bases...and insert. Once you’ve confirmed that the PCR was successful, mix together the vector and insert PCR ...Connecticut. Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without... of specific sequences for base pair overhangs, insert and vector purification, and, most importantly,...independent, it does not require purification of insert or vector products, nor does it require the use...In FastCloning, you PCR both the vector and the insert in separate reactions and then combine the products...procedure To start the FastCloning process, the insert and vector are first PCR amplified. The forward...

... the overhangs of the assembled inserts. After ligation of the insert into the vector, the BsaI site is...transformants by colony PCR confirmed the insertion of the Lambda insert at the same high frequencies as the...single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway... BsaI, and a DNA ligase such as T4 DNA ligase. Inserts, either precloned or in amplicon form (shown above...ordered, pre-determined manner. If the original insert religates into its original vector, it will subsequently... assembly: High efficiency and accurate single-insert assembly Intermediate 5- or 12-fragment assembly...assembly More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...

...depositing lab page contains all the plasmids from the same lab while the publication page contains all the plasmids...s plasmid number here. Gene/insert: This describes what has been inserted into the backbone, and provides...the gene/insert, this information can be found here. There may be more than one gene inserted into a backbone...associated, such as when a plasmid contains certain fluorescent proteins. Finally, for many plasmids you ... backbone, and each is given its own gene/insert section on the plasmid page. Tags are listed according...or C terminus of the transcribed and translated insert. If it is an empty vector with a tag, it’s terminus.... coli lab strain DH5a, but others may require strains such as ccdB survival (for Gateway plasmids).  ...