...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...

.... 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...

...our repository that express fluorescent proteins. ID Name Insert Antibiotic Resistance Purpose PI 17446...Plasmid Pooled Libraries . ID Name Description Genes/Insert Selection PI Human knockout pooled libraries 73178...with 1000 non-targeting controls. This backbone contains SpCas9. SpCas9 and 76,441 unique sgRNAs targeting...interrogate the genome in a variety of ways. Cas9 proteins can be guided to specific genomic sites by an ... ) or single-stranded breaks ( nicks ). ID Name Insert Function Antibiotic Resistance Activity PI 52962...and used as a transcriptional activator . ID Name Insert Antibiotic Resistance Activity PI 61422 dCAS9-VP64...barcoding library. Version 1 of the Celltag library contains 19973 barcodes to combinatorially index cells ...

...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,...

...PubMed . Direct identification of insulator components by insertional chromatin immunoprecipitation. Fujita... Locus-specific ChIP technologies consist of insertional chromatin immunoprecipitation (iChIP) and engineered...with engineered DNA-binding molecules such as TAL proteins and the CRISPR system consisting of a catalytically...s13104-018-3486-3. PubMed . Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative ...enChIP technology using recombinant CRISPR ribonucleoproteins. Fujita T, Yuno M, Fujii H. Genes Cells. ...system using recombinant exogenous DNA-binding proteins.Fujita T, Fujii H. BMC Mol Biol. 2014 Nov 27;15:26.../s12867-014-0026-0. PubMed . Identification of proteins associated with an IFNgamma-responsive promoter...

...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...

...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...

...(deletions or small insertions) or (B) span the genome-insert boundary (insertions only). The PCR product...dual) gRNA targeting 5′ exon or essential protein domains High-fidelity Cas enzymes increase specificity.... gene, such as generating a point mutation or inserting a tag Cas9 (or Cas9 nickase); Base editor Single...using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to generate...target exons that code for known essential protein domains. The benefit of this approach is that even non-... function when they occur in important protein domains. For gene editing experiments using HDR, it is ...targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using...

...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...

...to boost the research focused on Solute carrier proteins (SLCs). Browse the SLC plasmid collection....Plasmids Pooled Libraries Feedback Solute carrier proteins (SLCs) are the largest family of transporters ...associations. Plasmids This plasmid collection contains codon-optimized ORF sequences for human SLCs in...Gateway-compatible pDEST vectors. ID Plasmid Gene/Insert Industry PI Pooled Libraries CRISPR and overexpression... libraries target the solute carrier family of proteins. Library ID PI Description Human SLC Activation... CRISPR activation library targeting human SLC proteins. Human SLC Knockout Library 132552 Superti-Furga...Superti-Furga CRISPR knockout library targeting human SLC proteins. Mouse SLC Knockout Library 141436 Superti-Furga...

... 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...

...of interest, a medium copy RSF1030 origins or a low copy p15a origins should be used. Find further details...system to make singly or multiply phosphorylated proteins (Pirman et al., Nat Comm 2015). Improved phosphoprotein...improved ease of use when expressing recombinant phosphoproteins. These attributes make rEcoli XpS ( 192872 ...other plasmids containing high copy ColE1/PUC-like origins. Therefore, when choosing plasmids to express the...sequences corresponding to every previously observed instance of human serine phosphorylation ( Rinehart Human...phosphosite library with four different phosphobinding domains (14-3-3β, 14-3-3σ, the WW2 domain of NEDD4, and...68307 ) to generate the phosphosites with serine instead of phosphoserine (i.e. a “phosphorylatable” phosphosite...

...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...

...nerve cell function within the cell. Parkinson's Disease Parkinson’s disease (PD) is a chronic and progressive...More Fluorescent Proteins Find information on Addgene's collections of fluorescent proteins, including Markers...Differentiation Factors Antibodies Fluorescent Proteins Other Resources Neurodegenerative Disorders Neurodegenerative...neurodegenerative diseases include Alzheimer’s, Parkinson’s, ALS, and Huntington’s Disease. These diseases...with the mutated HTT gene will have huntingtin proteins with abnormally long polyglutamine sequences. ...with concomitant expression of aggregation-prone proteins with the iPSC toolbox . Lam et al. bioRxiv. 2022...A foundation dedicated to finding a cure for Parkinson's disease through an aggressively funded research...