...editing techniques. Inbred and outbred strains Mouse strains used in research can fall under one of two.... Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally ...Examples of outbred strains include Swiss Webster and Harlan (Hsd) National Institutes of Health (NIH) Swiss...broad classifications: inbred or outbred. Inbred strains share a common genetic background, which means ...limited but genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic...genetic diversity of the human population. Outbred strains of mice are useful for comparing dominant and recessive...Genetically engineered mice Mice can be engineered by inserting a targeting vector containing the gene of interest...

...Hospital  Brandeis University  Broad Institute  California Institute of Technology Cold Spring Harbor Laboratory...Laboratory  Dana-Farber Cancer Institute  Fred Hutchinson Cancer Research Center Memorial Sloan-Kettering...  National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK)  National Institute of Genetics...Rockefeller University  Salk Institute for Biological Studies The Scripps Research Institute St. Jude Children's...University and Medical Center  Weizmann Institute of Science Whitehead Institute for Biomedical Research Glossary...intellectual property, and to help requesting institutions safeguard themselves from undue liability. Before...materials? If so, you should always talk to your institution’s TTO or legal office first. Generally, Addgene...

...could be affected by the location of insertion. Lentiviral insertion can also disrupt the expression of ...Low Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual...Low Efficient and Stable Genome integration Insertional mutagenesis, residual expression piggyBac ... inefficient Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods...of iPSC generating methods due to the risk of insertional mutagenesis. Random integration also creates ...potential. Finally, retroviruses are prone to insertional mutagenesis and random integration creates variation...Addgene. Another drawback is that lentiviral vectors insert in different genetic locations in each transduced...

...generations of scFvs against two antigens that are impossible to generate scFvs against in vivo: antigens...the sequences for the variable domains of the selected scFvs are inserted into a vector encoding the antibody... are often the building blocks for engineering proteins. So let’s get acquainted with scFvs. What is an...variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that...scFv. While you might assume that the variable domains of an scFv would mirror that of an antibody (VL-linker-VH...antigens found in the body or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size...to produce scFvs in mammalian, yeast, plant, and insect cells, they are most often expressed from a plasmid...

...ankyrin repeat proteins, or DARPins, are genetically engineered small affinity proteins, around 14-15 ...antibodies. Instead, they are derived from a class of binding proteins known as ankyrin repeat proteins. DARPins...and heavy chains made by immune systems. But here, we're not interested in antibodies. Instead, we're going...engineered fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. ...synthetic binding proteins which use the Fibronectin type III domain as their scaffolding instead of the antibody...the ability to specifically recognize and bind proteins. They’re used in a number of research and clinical...these alternative reagents are created in the lab, instead of generated in vivo, they have higher reproducibility...

...  The best applications for dsDNA inserts include: large insertions or deletions, including selectable...note* not all linked strains contain the Lambda Red system).  In some of these strains, expression of exo... used to make an assortment of modifications: insertion and deletion of selectable and non-selectable ...of the experiment. dsDNA substrate is best for insertions or deletions greater than approximately 20 nucleotides... only a few base pairs.  dsDNA Substrate dsDNA inserts can be made by PCR using primers that amplify the... with 50 base pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically...not determine the direction of replication and instead design oligos that target both strands. One of ...

...high quality DNA that contains these elements. Be aware of endA+ strains Strains like Stbl3 have been ...incomplete. Protein expression strains are not good for cloning Bacterial strains adapted for protein purification...bacterial strain Differences between E. coli host strains can impact plasmid yield. Check the bacterial strain...an example). Look out for carbohydrates Certain strains, including HB101 and derivatives, release large...have previously reviewed many common lab E. coli strains on the blog, and also recommend this handy guide...for a more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture... be affected by the plasmid size, nature of the insert, propagation strain, growth conditions, and the...

...tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...recombinant proteins for one-step purification with glutathione, can also protect against proteolysis....one form of instability. Prokaryotes can also have a hard time folding eukaryotic proteins. You can get...101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection... biologists who need to overexpress and purify proteins can face any number of technical challenges depending...protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful protein... but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve disulfide...

... of posts explains how to critically assess the reported properties of fluorescent proteins, how to do...task. Many fluorescent proteins are available and the number of fluorescent proteins is steadily increasing...Before you start a new project with fluorescent proteins, the best advice is to try a couple of promising...do a head-to-head comparison of fluorescent proteins and how to make a well-informed decision on the best...data that summarizes properties of fluorescent proteins on websites. To make the best choice, the fluorescent...relevant conditions. The properties of fluorescent proteins are usually only examined under a few conditions...and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al...

...working with purified proteins in vitro, you can even use the same tag for multiple proteins but label each ..., V. W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research...protein of interest tagged with different fusion proteins for different experiments over the years — a green...been mutated to block its catalytic activity and instead become trapped in a covalent bond with its substrate... dyes provide many advantages over fluorescent proteins, opening up new applications.   Figure ...Griffin et al., 1998), this 15 amino acid peptide contains a cluster of four cysteines (CCXXCC, where X is... Michael Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR...

... yeast overexpression vector. This plasmid contains origins of replication and selectable markers that...the S. cerevisiae met deletion strains and then isolate transformed strains on media that lack uracil, but...function wide open. What if two seemingly similar proteins have acquired divergent functions due to mutations...research questions. The idea is straightforward. Instructors first select a multi-gene pathway to study and...and a reference organism where deletion strains are available. Then, staff or students clone similar sequences...overexpression plasmids. Students next transform deletion strains from the reference organisms with the overexpression...addition, a complete set of well-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae...

...Fellow in the Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is...Listen to this episode for insights into the process of getting an MD PhD, the inside scoop on developing new...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided ...mutant microbial strains faster than ever before. 29:54 - 31:25 : Nature's defenses against gene drives. ...Page. 2:00 - 3:55 : Alex introduces himself and explains why he decided to do an MD PhD - it's all in the...- 7:04 : Alex describes the MD PhD process and explains why he loved the first 2 years of medical school.... 13:22 - 22:07 : Alex's projects at the Wyss Institute - New CRISPR tools and the importance of moving...

...binding proteins in as little as 3 days. Abil et al., 2014 show that you can fuse custom PUF proteins to a...you’re an academic researcher at a nonprofit institution, you can order plasmids covering a wide range...expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression vectors...antibiotic efflux pumps and ankyrin seven-repeat proteins: Sjuts, Hanno, et al. "Molecular basis for inhibition... from affecting its translation initiation and instead makes initiation heavily dependent upon the core...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium... "Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs." PloS one 11.2 (2016): e0149483...

...with dimerizing fluorescent proteins is illustrated. 1) Membrane proteins fused to FPs that dimerize can... National Institutes of Health and he is currently an Associate Professor at Albert Einstein College of...-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429... Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal...(i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs is to visualize...Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors and transporters, are integrated... to an obligate dimer or oligomer Several cell proteins normally self-associate into homodimers or even...

...sorting or FACS (2). By analyzing single cells instead of the population as a whole, scientists gain statistical...Techniques is Best for Me?  An overview of the instrumentation A flow cytometer combines three systems to ...receptor are stained with a fluorescent antibody against the receptor and analyzed on a flow cytometer. ...cells pass through the path of a laser and the instrument collects information about the cell’s size, complexity...targets. If you are planning on using fluorescent proteins for your experiment and are unsure which to choose...Fluorescent Protein Should I Use? However, fusion proteins tag your protein of interest with a fluorescent...C-terminus of the protein. One downside of fusion proteins is that the tag can alter the structure of the...

... Georgeta, and Justin W. Taraska. "Imaging proteins inside cells with fluorescent tags." Trends in biotechnology...Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry! The discovery... their own. Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive...budgets are a widespread issue with fluorescent proteins—even the thriftiest cell biologist can feel like...brighter and more photostable than fluorescent proteins, providing a straightforward way to improve the...combined with the ease and specificity of fluorescent proteins. In-cell labeling strategies The majority of ...tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have...

...occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968; Bazaral & Helinski, 1968), in which the plasmid was ...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... of plasmids, taking many forms (Levene, 2009; Higgins & Vologodskii, 2016). Plasmid multimerization occurs...to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro...et al., 2009). Since multimers have additional origins of replication compared to monomers, they are also... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can.... If the presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the...

...Mutations Insertions, deletions, point mutations Insertions, deletions, point mutations Insertions, deletions...-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the ...recombineering proteins Red/ET from a temperature-sensitive plasmid. Homologous recombination then joins the linear...successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer synthesis is more expensive...making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly...the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency...colonies carrying the unmodified plasmid. For insertions, it’s possible to use the same technique just...

...REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic domains, as well as the CTD (...recognition). The NUC lobe contains the DNA scissors: the HNH and RuvC domains, which are named for similar...the guide RNA. Unlike the cleavage domains, the recognition domains—including the CTD and the entire REC...similar nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs ...lobe—do not structurally resemble any other known proteins; they are completely unique to the CRISPR system...Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs to, and their roles...pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange;...

...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing two ...research requires a close examination of specific proteins within a system. This can be pretty complicated...given that a single cell has tens of thousands of proteins functioning in a variety of ways. How do scientists...from a complex mix using affinity chromatography against an amino acid sequence, or tag, on the protein....of methods can be used to successfully isolate proteins, this blog will focus on one of the most common... as Ni2+ or Co2+ (Hochuli 1988), and can elute proteins with up to 95% purity (Janknecht 1991, Hochuli...itself. Next, a series of wash steps removes excess proteins and other contaminants. The protein of interest...

... and analysing the insecticidal potential of Cry proteins in order to manage insect pests and to support...The recombinant Cry proteins will then be screened for toxicity against local insect pests. By developing...the identification of plant compounds and insecticidal proteins to the development of renewable biotechnology...desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc.), Nong Lam University...developing biological agents such as insecticidal proteins, the use of chemical pesticides may be avoided or...system pDawn to clone and express newly identified proteins and enzymes. These compounds will later get tested... nutritionally and therapeutically significant proteins. In this project she aims to create transgenic...

... of proteins captured on magnetic beads (Arimura et al., 2024). In MagIC-cryo-EM, target proteins (expressed...adding Late Embryogenesis Abundant (LEA) proteins, small proteins that naturally adsorb to the AWI, can ...editor installs the recombinase “landing sites,” then the recombinase handles the installation of the ... DNA-targeting proteins, so the team created versions using dCas9, zinc finger proteins, and transcription...for enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...https://doi.org/10.1101/2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid plunge...

...Broad Institute focuses on proteomics: developing and applying new technologies to quantify proteins in ...locus and labels the proteins in its vicinity with biotin. The biotin-labeled proteins can then be isolated...electron-rich amino acid side chains (e.g. Tyrosine) with biotin. These biotin-labeled proteins can them be analyzed... anti-GFP nanobody is able to bind fluorescent proteins derived from A. victoria the functionalized nanobodies...: 29915061.        A new method to identify proteins occupying a specific genomic locus: Caspex  Article...group, developed a CRISPR-based approach to detect proteins that associate with specific genomic loci. He ...engineered peroxidase, (APEX) that labels proximal proteins with biotin as an affinity tag. This ‘Caspex’ ...

... Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology...to effector domains such as transcriptional activators or repressors. These fusion proteins retain the .... For instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that contains an RNA..., or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary..., in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding... full-length gRNA, native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands complementary... a regulator of gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic...

... measurement that is unaffected by intervening proteins, lipid bilayers, organelles or other cellular ...et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest are fluorophore...resulting from nonspecific donor labeling. Fluorescent proteins (FPs) are the gold standard for genetically-encoded...remember that, if you make an FP fusion, you are inserting about 20-25 kDa of protein mass into your protein...protein and so structural changes due to this bulky insertion are inevitable. Most FP fusions for FRET studies...can affect the structure or function of certain proteins. Rather than shove a bulky FP into your protein...pair, a researcher can lure the ribosome into inserting a fluorophore-tagged amino acid at a stop codon...

... Vector Database. Step 2: Insert name  Include information about the insert in your plasmid name. This...position to the insert. Example, if you have a Flag tag on the N-terminal of your insert, you would list...fused to the C-terminal of your insert you would list it after the insert. pBACKBONE-Flag-hGene-EGFP Other...Other considerations  If your insert contains a mutation or modification, this should be included in the...versions of the insert are often denoted by "wt" either directly before or directly after the insert name.   ... quickly and easily identify what your plasmid contains and other important information. Tip: A lowercase...antibiotic resistance, b) the promoter that drives the insert, and c) any other selection markers (for use in...

...Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied in a long list of...light. To do so, she generated fusion proteins in which Dronpa domains flanked either side of an enzyme’s...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable...researchers are finding new ways to apply fluorescent proteins everyday.  The late Michael Davidson and Roger...for their creativity in the use of fluorescent proteins and the potential for their work to enable future...that list by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off... exposure caused oligomerization of the Dronpa domains thereby occluding the enzyme active site and shutting...

...for tDNA insertions into the plant’s germline Selecting for seeds that have the tDNA insertions (usually...have no control over how many insertions occurred. Having too many insertions can cause artifacts from over-expression...control over where your construct will be inserted. If your insertion occurs in a coding region, it may cause...transcriptional and translational dynamics of genes and proteins in living plants, to complementing mutant phenotypes... process occurs in nature; Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes...and generating N and C terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or...perform the transformation not long after flowering begins. If your plants begin to flower before your constructs...

...only indicates the presence of AN insert, not necessarily YOUR insert. Any cloning artifact that disrupts...able to choose those that contain plasmid with an insert? Blue-white selection is a widely used method to...beginning. The well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme...indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-indigo). So far,...an orange wedge in the figure on the left) and inserted it into a plasmid, creating an α-complementation...representative plate to the right, colonies with an insert-containing plasmid have a non-functional β-galactosidase...control: Transform the backbone plasmid without insert. All colonies on this plate should be blue, indicating...

...Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins, and channelrhodopsins...of optogenetics begins almost 50 years ago with the discovery of microbial rhodopsins, light-sensitive...light-sensitive proteins that absorb photons for energy conversion or the induction of intra- or intercellular signaling... et al., 1984; Harz and Hegemann, 1991). These proteins control the flow of electric charges across cell...light (Zhang et al., 2011). What do microbial rhodopsins and neurons have in common? Neuronal stimulations...when hit with the correct wavelength, microbial rhodopsins enable ions to flow across the axon’s membrane... 2012). For example, blue light activates channelrhodopsins that, in turn, trigger neural excitation, ...

...system is likely applicable to many types of proteins. For proteins with a modular structure, constructing ...Other proteins may also be utliized as scaffolds, including the set of red fluorescent proteins. The T-DOGGs...researcher Jonathan Tang wanted to express a variety of proteins in single cell types in the mouse, but knew it...single heavy chain, and they are small and stable inside cells. Nanobodies binding GFP were designed in ...showed that nanobodies could be fused to other proteins, and these fusions retained the ability to bind... Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP....similarly to GFP, but commonly used red fluorescent proteins dsRed, mCherry, and TdTomato do not induce transcription...

...series, proteins are overexpressed from a plasmid construct, most often in special E. coli strains designed...difficult-to-fold proteins. In addition, P. pastoris has the capacity to glycosylate proteins and may recapitulate...difficult-to-purify membrane proteins. pCri vector pCri-13a, designed for use in B. subtilis, contains the MISTIC (membrane-integrating...MISTIC drags tagged proteins into the membrane and may improve expression of proteins that natively localize...the most stressful lab activities. Working with proteins requires a substantial amount of properly folded... your protein. Structural information for many proteins is easily accessible in the RCSB Protein Data ...and stability. TRX and signal peptide tags help proteins with disulfide bonds fold properly. If a TEV protease...

...about the origins of some of the common techniques or tools you use in the lab? Take for instance, the commonly...only be added to the C-terminus of proteins of interest and proteins tagged with it needed to be chemically...tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila ...we explore… the life and times of the myc tag.  Origins of protein tags For about as long as the disciplines...antibodies as tools allowed us to detect and capture proteins of interest to better study them in vivo and in...they landed on was to recombinantly append their proteins of interest with unrelated peptides that already... molecular biology. And the Myc1-9E10 antibody remains a trusty antibody partner; as I write this, there...

...working with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often change shapes... of the proteins to the microtiter walls for ELISAs and a more accurate separation of proteins by mass...out of a complex mix of proteins that have been extracted from a cell, instead of when it is attached ...browse the collection, you'll likely find some proteins of interest for which you have several antibodies...the protein, the antibody can't bind to it. As proteins change configurations or binding partners, the...as ELISAs and Westerns, frequently denature the proteins as part of the sample prep. This involves using...epitope accessibility naturally, by binding to other proteins, changing locations, or even just as part of performing...

...utility of PhoCl in a variety of settings. For instance, inserting PhoCl in between a protein and a tag, such... activity. In addition, proteins can be “caged” by fusion to specific domains thereby rendering them inactive...demonstrate that structurally defined domains can be swapped onto other proteins to confer functionality. In Narayan..., with the zinc finger domains of TZAP showed that the TZAP zinc finger domains were sufficient to localize...and molecular processes via genetically encoded proteins. PhoCl is a new member of this class that works...NLS or NES, enables control over localization. Inserting PhoCl between an enzyme and an inhibitor domain...of new RFP variants The toolbox of fluorescent proteins (FP) for cellular imaging is constantly expanding...

...viral proteins in human cells and used these viral “bait” proteins to fish for human “prey” proteins that...UCSF). Our approach is to discover key human proteins that can be targeted (hopefully by existing therapeutics...the virus. As a result, we identified 332 human proteins that the virus interacts with in cells. Because...Because viruses rely on the help of human proteins in their lifecycle, identifying these potential human helpers...better, more than half the drugs hitting these proteins are FDA-approved or in clinical trials or preclinical...Paris and New York. We want YOU to join the fight against coronavirus We are just at the beginning of understanding...understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T cells in our first...

...pairs long and contains a single origin of replication (oriC), where initiation begins. This process relies...recruits host proteins to the ori to initiate replication. At high copy numbers, RepA proteins form complexes...when they depend on the host cell’s initiation proteins to start their own replication. In general, these...bound, including at the low-affinity sites, the proteins work cooperatively to separate nearby AT-rich ...DnaA boxes are occupied by ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created...to control DNA replication. A variety of other proteins also contribute to this regulation in different...chromosomal replication and balance population growth against sustainability. Plasmid DNA replication and regulation...

... host replication machinery. For instance, the ori typically contains iterons that serve as recognition...plasmid harbors distinct origins (ori1, ori2, ori3) and replication initiation proteins (RepA, RepB) necessary...such as Rep or other initiator proteins, they do not require host proteins for replication and can maintain... are: The plasmid origin of replication (ori) contains structural elements that are versatile and adaptable...recognition sites for replication initiation proteins. These iterons vary in their sequence and number and can...oris interact with various RNA polymerases and proteins from bacterial hosts. In a plasmid with multiple...has its own replication initiation factors and proteins independent of the host. Host range depends on...

...Controlling gene expression through gene sequence - Proteins that are encoded by codons with low abundance ...charged tRNAs may be produced at a lower rate than proteins encoded by highly abundant, charged tRNAs. For...secondary structures in 10 closely related yeast strains. Adaptation to changing conditions - Organisms ...varied codon usage, an organism can change which proteins are highly expressed and which are poorly expressed... problematic for researchers trying to express proteins in heterologous hosts. If you simply amplify a... a gene of interest from the human genome, for instance, it may not express at all in E. coli (you can...transcript resulting in the production of nonfunctional proteins and protein fragments respectively. Solving the...

...Unfortunately, using proteins as bait has limited utility for identifying novel RNA-binding proteins that interact...which host proteins interact with viral RNAs. RNA viruses, like Zika, often trick host proteins into binding...from BioID and allows a researcher to identify proteins that bind an RNA motif of interest in living cells...RNA-protein interactions by cross-linking RNAs and proteins with UV light, followed by an antibody pulldown... hours to sufficiently biotinylate interacting proteins, which results in poor spatiotemporal resolution...promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within ~10 nm of the bait, which for ...labeling methods, RaPID cannot distinguish between proteins that directly or indirectly interact with the ...

...possible number of target proteins.   The exception to this rule is Western blots. Instead of using signaling...secondary antibody generated in goat against rabbit immunoglobulins. References and Resources More resources...molecule is conjugated to the secondary antibody instead of the primary antibody. Since multiple secondary... molecules are represented in light green; and proteins are represented in orange. Created with BioRender.com...  Secondary antibodies are antibodies generated against antibody isotypes from a specific species, such...possible to get secondary antibodies generated against isotype subclasses, such as mouse IgG2 or IgG4....varies across applications. Western blots, for instance, almost always use the indirect approach, while...

...backbone with one insert, and can use common primers to sequence from the backbone into the insert. If the insert...and identity of the insert are correct. In other cases, we deal with multiple inserts or internal mutations...(such as LTRs or ITRs), hairpins, or strong terminators. Detailed instructions on other factors to consider...DNA samples every week. Here we will provide an inside look at the steps we take to verify the identity...information for a given plasmid are the backbone, inserted gene/functional mutations, antibiotic resistance... of certain plasmids, such as special growth instructions (temperature, strain, media supplements), suggested...most cases at Addgene, we check what we call the “insert,” (the gene or genetic element cloned into the ...

...  The lentiviral transfer plasmid encodes your insert of interest, flanked by long terminal repeat (LTR...portion into the host genome. The packaging plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for...AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated ...integrated into the host genome via the viral proteins packaged during production (read our blog post on the retroviral...functioning integrase, the enzyme responsible for inserting viral DNA into the host genome. Without a working...into a large complex of both viral and cellular proteins and trafficked to the nucleus. Since the integrase...IDLV over a lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors tend to integrate...

...attached to self-labeling proteins. Some common examples of these self-labeling proteins would be SNAP-tag® ...cells or specific proteins, can influence the choice of fluorescent dye. For instance, the cleavage of ...fluorescent proteinFluorescent Proteins 101: A History of Fluorescent Proteins  ...interiors to many inquisitive scientists. Seeing inside these specimens has offered illumination on biochemical...an antibody to detect various targets, such as proteins, nucleic acids, or glycans. Fluorophores can also...several advantages to using these self-labeling proteins over traditional fluorescent protein tags, such...stability of fluorescence over time is crucial. For instance, the fluorescence of fluorescein isothiocyanate...

...ELISA, purified proteins or the experimental sample are bound to the wells of a plate instead of an antibody... of proteins in an ELISA well As mentioned above, the measurement of the concentration of proteins in ... experiments that require scientists to detect proteins in their samples. One technique that relies heavily... Immunosorbent Assay. ELISA are used to detect proteins within a 96- or 384-well, flat-bottomed plate....below). The sandwich ELISA is best used to detect proteins for which multiple antibodies that recognize different...it can also be used to characterize antibodies against the antigen bound to the plate. Figure 3...is coated onto the wells of the plate. However, instead of detecting your protein using an enzyme-conjugated...

...easily visualize single molecules and newly formed proteins.  Limitations with GFP in imaging  While GFP ...GFP has been an immensely powerful tool to study proteins in vivo, it does have some limitations. GFP has...impossible for scientists to use GFP to image nascent proteins. In addition GFP tags are large, dim, and permanently...epitopes and scientists can use these to tag their proteins of interest. Antibody based probes, such as antigen-binding...fluorophores, and can bind and light up epitope tagged proteins in vivo. These probes however can be expensive...living systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat...towards a HA epitope from the CDRs.  To visualize proteins of interest tagged with the HA epitope, the lab...

...recombined vector backbone contains the antibiotic resistance gene (AmpR) and origins of replication (ori and...strain While common E. coli strains like DH5-alpha are recA-, some strains have been engineered to further... interest (aka the viral genome) and the other contains the bacterial selection marker and origin of replication...further reduce recombination. Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce...For a more detailed comparison of these cloning strains, browse the table of common gene mutations in our... our Plasmids 101: Common Lab E. coli Strains blog post. Check yourself before you recA yourself. Optimize...concentration of antibiotics (ie. 50 μg/mL Amp instead of 100 μg/mL Amp), or using more nutrient rich ...

...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 ...