...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...

...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...

...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...

... 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...

...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 ...

...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...

.... Inhibitory opsins are usually light-driven pumps that pass ions (Cl-, H+, Na+) against the concentration...Light-gated ion channels Light activated ion channels (rhodopsins) either excite or inhibit neuronal activity upon... of light (Vierock et al., 2021). Step-function opsins are chimeric ion channels that turn on with a light-pulse...are many different opsin (natural and engineered proteins responsive to light in combination with retinal...activation inhibitory (ArCH) proton pump passing ions against the concentration gradient. Created with BioRender.com...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate...partners such as light, oxygen, and voltage (LOV) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2...

...fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins to make ... imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs) can be problematic...fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging tools but their use is limited...tissues. To avoid absorbance of heme-containing proteins in whole animals, light at or above 600 nm wavelengths...fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited by Nluc emission and the lab has...maturation time compared to similar orange fluorescent proteins. In their work, the authors noted how CyOFP1 could...of the Enhanced Nano-Lanterns to other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase...

... 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).  ...

... 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...

...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...

...well — namely that they can cause cancer via insertional mutagenesis (which HIV does not do). Furthermore... yet retroviruses are RNA-containing viruses  (Robinson et al., 1965)! You may be beginning to realize...the fact that the virus must have its structural proteins expressed from a reverse-transcribed piece of ...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... The RT protein is made by a separate set of instructions in the same producer cells as the virus. Because... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...

...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...optimizing strains with low efficiency.       Sheng Yang obtained his Ph.D. from Shanghai Institute of Biochemistry...2015). In addition to the pMB1 replicon, pTargetF contains the sgRNA targeting the genomic site of interest... system.   Figure 1: The editing process begins with the addition of the sgRNA sequence in pEcgRNA...method was much simpler and took only ~32 hours instead of ~60 hours. We successfully tested the pEcCas...precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade and pCutamp...

...distances. For instance, how much force can be generated by conformational changes in proteins? How can information...produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance...rupture E. coli spheroplasts to release encapsulated proteins. Video by ACS. For a more cartoony explanation...rearranging into different polymerization states. Instead, R body monomers respond to pH changes by undergoing... variety of conditions. They are composed of 4 proteins encoded in a single operon and were first expressed...

...Antibodies are composed of two light chains and two heavy chains. The ends of the branches contain the...distribute monoclonal antibodies against popular fluorescent proteins (GFP and YFP) and protein tags (...binding specificity allows scientists to target proteins of interest for labeling, quantification, purification...cell lines. The DSHB was created by the National Institute for Child Health and Human Development (NICHD)...distribution funds our operation. Antibodies are glycoproteins that are part of the humoral immune system. ...hybridoma lines, each producing a monoclonal antibody against a target antigen. This contrasts with polyclonal...popular monoclonal antibodies include those directed against human transcription factors such as PAX7, PAX6,...

...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming... benefit of working with surface proteins is also that such proteins are easily studied in living cells...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest...screen, each gene is not targeted by only one gRNA, instead many different gRNAs are used for each gene. At...molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different contexts.... Thus, a screen can be used to target surface proteins to determine which contact points are involved... used to interfere with the identified surface proteins, and such antibodies could thus be interesting...

...pull-down assays: Epitope tagging provides a solution Proteins - the workhorses of the cell – never work alone.... It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform...complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes... mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments...for each target protein. To study 20,000 human proteins, this would be time-consuming, expensive, and ...biochemical “epitope tag” such that these tagged proteins can be immunoprecipitated using a standard tag-specific...assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed...

...cells, live cells, my marker stains of interest). Let’s double-click inside our gate and proceed to the...present that vary in cell type, size, and marker proteins. But even if you are analyzing a homogenous cell... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage... the magnitude of FSC, SSC, and all fluorescent stains. The cytometer's software will allow you to adjust...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...where multiple cells were clumped together. (B) Instead of FSC, one is free to use SSC for a single cell...separation pattern of positive vs. negative signal remains the same (Figure 5).   A.                                                                                                        ...

...effectively biotinylate AviTag expressing proteins in bacteria, yeast, insect and mammalian cells. Check out Kay...about in vivo biotinylation of bacterial fusion proteins Biotin is an essential vitamin for life Although... immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns...help with the specific elution of biotinylated proteins. For help determining which biotinylation reagent...Biotinylation Reagent Selection Tool. AviTag and BirA Proteins can also be biotinylated by exploiting the specificity...Kay et al., 2009 for methods on biotinylating proteins with BirA. You can find plasmids that contain ...Scientists have commonly used this method to attach proteins or polypeptides to a solid substrate (Holden and...

... 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific...anhydrotetracycline (aTet), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting... point mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is.../pEcgRNA, allows it to be used in more E. coli strains including the popular BL21(DE3) (Li et al., 2021...tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of... both systems, custom spacers/gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid...plasmid can be also linearized with XbaI to insert extra elements, like a repair template, using Gibson...

...refers to 3 proteins (H-Ras, K-Ras, and N-Ras) encoded by a family of proto-oncogenes. Ras proteins are special...” shape and lack of “druggable” domains. In other words, Ras proteins naturally lack areas that are accessible...Resources National Cancer Institute: Cancer Statistics Page National Cancer Institute: Ras Pathway v2.0 Page...products. Proto-oncogenes consist of a group of genes/proteins that, when expressed, normally encourage cell ... right places. Checks and balances: protecting against cancerous cells Luckily, there are several safety...not ideal environments for many cell types. From proteins to pathways - Ras as a model When mutations do...different cancers and identifying the roles of key proteins involved, with the goal of being able to discriminately...

...most famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well, both natural...you. We’ll cover the common terminology for the proteins, DNA, and RNA used in CRISPR.   Figure...tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed...spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs...Cas: Different CRISPR systems involve different proteins, but many of them are called Cas, short for “CRISPR-associated...natural and engineered. What these proteins all have in common is the ability to use an RNA guide to target...Target strand: The strand of foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand...

... 13(4):602-606. Brian, D. 1996, Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  ...their trainees’ very way of thinking too. Albert Einstein might have put it best when he stated, “the value...metabolic pathways (precisely the type of exercise Einstein might have resented!). In 1997, Joseph Provost...structure, function, and interaction with other proteins.   It is important to note that research ...Kimball et al., 2020). Benefits and Challenges CUREs inspire students to take ownership of their work and become... him a more effective motivator (“be like Tony Robbins!” he encourages faculty mentors.)  Collaboration..., Provost partnered with colleagues at other institutions to form a support network called the MDH CUREs...

...is fused to the DNA backbone that contains a target sequence against the fluorescent reporter in an inverted...single hairpin dsRNA is produced that contains sequence against your gene(s) of interest and the reporter... and Biotechnology from Worcester Polytechnic Institute. What is RNAi? The loss-of-function (LOF) experiment...downregulation, or “silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and... is facilitated by DNA constructs that enable insertion of ~400 bp complementary to your gene target as...whereas shRNAs are exogenously derived. Importantly, instead of the assortment of siRNAs generated from a long...outside of limited, single cell protoplast analysis. Instead, microscopic plants must be visually identified...

... enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains to cleave target DNA... HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a molecule results in...SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent the limitations..., and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a...form of “bait” to identify new CRISPR-associated proteins within the bacterial genome. From this analysis... DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent with known roles for HEPN...Comparison on Common CRISPR Enzymes Name  Enzymatic Domains Guide RNA Target PAM (PFS) Cleavage Mechanism ...

...recombinant proteins a cinch. This is because it doesn’t secrete its own endogenous proteins, making the...affordable, and it grows fast to boot. The yeast strains S. cerevisiae and S. pombe have dominated the research... between two of the most frequently used yeast strains: S. pombe (fission) and S. cerevisiae (budding)...established in yeast, including a widely used Parkinson’s experimental model. In fact, approximately 25%...differences between K. phaffii and other yeast strains.     S. cerevisiae S. pombe K. phaffii...and bears more similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii...was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries as...

...users can submit new fluorescent proteins, add data to existing proteins when missing, or edit inaccuracies... specific protein (for instance: EGFP).  Users can “favorite” certain proteins, or create custom protein...recognized by the 2008 Nobel Prize, fluorescent proteins (FPs) have become one of the most indispensable... one might require when making such a decision remains spread across literature in publications that introduce...community resource for information about fluorescent proteins.  It was designed with the goal of encouraging...Protein Properties provides a master list of all proteins in the database, and the Interactive Fluorescent...moderate your own version of fpvis.org with the proteins of your choosing!), and they can be downloaded...

...more fluorescent proteins are made. As the bacterial replicate, fluorescent proteins will be diluted out...excited to see the many creative uses of fluorescent proteins. We’re happy to announce the three winners of ...by fluorescence-activated cell sorting into four bins based on their eGFP:mCherry ratios. Genomic DNA ...response to warfarin. “These novel methods will be instrumental in interpreting human variation at large scales...making personalized medicine a reality,” Chiasson explains. Elizabeth Unger: Developing a serotonin sensor... sensor. However, there are no known bacterial proteins that bind serotonin. Unger redesigned the binding...patient and rapidly test the variants in vitro against different drugs and dosages.  Michal Bernach: Developing...

...candidates for further development. They inserted an octet of MS2 hairpins into the gRNA tetraloop and went through...-head against CRISPRainbow. Both tools were targeted to the FBN3 intronic repeat which contains 22 copies...the genomic loci of interest. Pairs of identical hairpins within one gRNA will result in a blue, green, ...red readout. But gRNAs containing two different hairpins will produce a secondary color (yellow, cyan, ...hairpin combination. A gRNA containing all three hairpins produces a white color. While CRISPRainbow allows...CRISPRainbow labeling. Two MS2, PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors...induce its fluorescence. They found that aptamer insertion in the gRNA tetraloop (a four base hairpin loop...

...majority of Type VI-D loci contain accessory proteins with WYL domains (named for the three conserved amino acids...domain-containing proteins may be broader regulators of Cas13d activity. This property makes WYL proteins an intriguing... near a putative effector nuclease. The Cas13d proteins they identified have little sequence similarity...Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily. Yan et...advantages, including Cas13d-specific regulatory proteins and high targeting efficiency, both of which are...intriguing counterpart to anti-CRISPR proteins that negatively modulate the activity of Cas enzymes, some...specific editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann...

...possible to generate antibodies against many types of molecules including proteins, lipids, and carbohydrates... post we’ll focus on antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas...hybridomas come from animals that were immunized against the target protein. Animals are first injected ...special medium called HAT. This medium selects against unfused myeloma cells and antibody-producing B ... will exhibit off-target binding to unintended proteins. This is because monoclonal antibodies recognize...amino acid sequence will be shared across other proteins. Conversely, polyclonal antibodies - which are...recognize epitopes that are also present in other proteins. Because of the specificity of monoclonal antibodies...

...podcast segment The human proteome contains a breadth of modular domains that facilitate protein-protein ...for RNAi. The Vellai lab’s new plasmid, T444T, contains T7 terminators adjacent to the T7 promoters to...TurboID and miniTurbo Article contributed by Eric Perkins  Listen to the TurboID and miniTurbo podcast segment... study of the distribution and interactions of proteins within these structures. Enzyme-catalyzed proximity...distribution and interaction networks of specific proteins in living cells. Until recently, the enzymes most...APEX2 and BioID. APEX2 is fast, tagging proximal proteins within minutes, but requires H2O2, which can be... tailored for the detection of other norovirus strains and even other viruses by modifying the sequence...

... This means recombinant proteins can aggregate or precipitate out as insoluble granules. We overcame this...decidedly uncomfortable 45°C). Their more adventurous cousins are the extremophiles - the legends who not only...of processes. The food processing industry, for instance, employs psychrophilic enzymes for milk processing...enzymes for the host The expression of recombinant proteins for industrial or research purposes requires stable... standard conditions. However, one of the many proteins which are not compatible with regular mesophilic...temperatures suitable for the expression of psychrophilic proteins. The primary problem we wanted to overcome was...folding and expression of recombinant psychrophilic proteins. As the figure below shows, E. coli expressing...

...available here for free!) contains strategies, hints, anecdotes, and insights. After I wrote the first ... at the National Institutes of Health, was an Associate Professor at Albert Einstein College of Medicine...Hughes Medical Institute. His interests include the quality control of secretory proteins in the Endoplasmic... and mentoring to several people that found my insights helpful. All went on to get faculty positions ...specific questions and experiments. Nobody EVER complains that a seminar was too easy to understand. Make... Distinguishing correctly folded and misfolded proteins is essential for human health. Failure to do so...terms. "Implications for human disease" is vague. Instead, consider "A mechanistic understanding of this ...

... HIV so that infected cells will produce viral proteins and die, either through cytotoxicity or the immune...transcription, but with two additional activation domains (MS2 and p65) to enhance transcriptional activation...expression. In cell lines that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating ...cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the HIV-1 5’ and...Importantly, no off-target effects were detected. Kaminski et al. observed no negative effects on cell viability...up to 7 mismatches to the gRNA target sequence. Kaminski et al. next tested their procedure in HIV-1 infected...expanded, and infected with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly ...

...composed of four peptide chains (two identical heavy chains and two identical light chains) each with a constant... The chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...are composed of just the variable domains of the heavy and light chains of an antibody. Arguably not a “...chain and one light chain. When assembled, the four chains make a Y-shaped protein that can be divided into...portions of the constant regions of the two heavy chains. In the lab, this is the region that determines...fragments are produced by proteolytic digestion that retains an antibody’s disulfide bonds within the hinge ...the fragment could bind not only the endogenous proteins you are trying to block but your primary antibody...

...endogenous proteins is an exciting strategy, it is difficult to apply this technique to oncoproteins that lack..., a postdoctoral fellow at Dana-Farber Cancer Institute. Targeted protein degradation In the Bradner and...synthesize compounds that enable selective removal of proteins-of-interest from the proteome. Rather than inhibiting...degraders” recruit the proteasome to destroy targeted proteins. We previously developed small molecule degraders...that achieve selective degradation of endogenous proteins (notably, BRD2/3/4, CDK9, TRIM24, FLT3, BTK, and...linking small molecules that bind these target proteins to other small molecules that bind an E3 ligase... molecule) and expression of FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation...

...these three kinases, photoswitchable Dronpa domains were inserted at sites homologous to the locations they... other kinases and proteins. Zhou et al suggest that more photoswitchable proteins could potentially be... be created by inserting pdDronpa domains into loops of a protein of interest. If you plan to try this...ninja term for vanishing, and pa= photoactivation) domains on either side of the active site of a constitutively... Learn More about Photoactivatable Fluorescent Proteins Control Protein Expression with Cre-lox Control...reporter plasmids Find photoactivatable fluorescent proteins in empty backbones in the Michael Davidson Collection...Photoswitchable Fluorescent Proteins ...

...transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the Wei Leong Chew Lab...repair proteins fused to CGBEs The David Liu lab also experimented with adding different proteins to the...approach to improving C → G editing. Instead of fusing DNA repair proteins to the base editor, they added the...inhibit BER during cytosine-to-uracil editing, and instead introduced its opposite, a uracil DNA N-glycosylase...excising the freshly edited U bases — but E. coli instead filled those abasic sites with A's, producing C...Mycobacterium smegmatis, termed UdgX, they found three proteins whose fusion improved the CGBE: DNA polymerase...(RBMX) (Koblan et al., 2021). Several of these proteins had additive effects when multiple were included...

...fluorescent proteins system for Caenorhabditis elegans By Jennifer Tsang Fluorescent proteins (FPs) traditionally...Tools for labeling excitatory inhibitory synaptic proteins Fluorescent protein system for C. elegans Fluorescent... Labeling excitatory and inhibitory synaptic proteins with FingRs By Jason Nasse Since the pioneering...neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin intrabodies generated...FingR constructs target the synaptic scaffolding proteins PSD95 and gephyrin to label excitatory and inhibitory...neuron, the FingRs bind to the endogenous synaptic proteins allowing them to localize to the synapse and minimize...combining vectors expressing different fluorescent proteins targeting inhibitory or excitatory synapses or...

... process. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each ...that bind to MS2 proteins. These MS2 proteins then recruit additional activation domains (HS1 and p65). ...transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems...method is its construction: it relies on antibody chains, which are relatively large and are not expressed...epitope tag of SunTag with the P65-HSF activation domains used in SAM. This method has been shown to activate...Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated...Kowal EJK, Buchthal J, Housden BE, Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators...

...images of proteins. But they are still flat. To truly appreciate the 3D structure of these proteins, it is...Molecular Designs, we create physical models of proteins using 3D printing technology. The tagline for ...to create stunning, interactive images of their proteins. Since the 1980’s, journal articles have been ...structure? We have been creating physical models of proteins for researchers for the past fifteen years. On...expressed purpose of creating physical models of proteins and other molecular structures for use in professional...Under the guidance of a trained educator, these instances of focused attention become magical teaching moments...their research while creating physical models of proteins that are being investigated in their labs. Soon...

...elements anymore. The kit contains sequences for several different fluorescent proteins. Visualizing fluorescence...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like an... between plants and their pathogens. As Patron explains it, her work has always been focused on gene transfer...Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and enable ...chlorophyll, so we looked at newer fluorescent proteins that get around that problem. MoClo is really ...straightforward really and we’ll be producing an instructional video soon to make it easier for people to use...for plants? Patron: Our lab and others at the Sainsbury are working on a lot of CRISPR tools for editing...

...forces its insect host to dive into water. There, the worm emerges to find a mate, leaving the insect dead ...infected grasshoppers expressed different proteins in their brains compared to uninfected grasshoppers, including...Plasmid pumpkins, team costumes, and spooky science stories. Yes, please! At Addgene, we love finding...addition to the Halloween contest, we’ve also carved pumpkins (yes, Blugene made an appearance there too) and...base of the tongue where it pierces the tongue and drains all the blood out. Once the blood is depleted, ... the tongue atrophies and falls off. The louse remains becoming the fish’s new “tongue” (Brusca and Gilligan...Spinochordodes tellinii‘s larvae makes its home inside grasshoppers and crickets. When it’s ready to transform...

...lentivirus, but instead is contained by a capsid which is comprised of the viral proteins VP1, VP2, and ...Maday et al., 2014). It is currently unclear which proteins facilitate transport of neuronal tracing compounds...peroxidase (HRP; Kristensson and Olsson, 1971), plant lectins (wheat germ agglutinin WGA, Schwab et al., 1978...iontophoresis (applying small electric currents via inserted electrodes) may be applied, but these methods ...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...methods such as electrophysiological recordings. The origins of these methods are naturally occurring viruses...engineered to have differences in their capsid proteins. For a brief overview see Addgene’s AAV guide....