We narrowed to 1,049 results for: ins

...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...

...attached to self-labeling proteins. Some common examples of these self-labeling proteins are SNAP-tag® and HaloTag...cells or specific proteins, can influence the choice of fluorescent dye. For instance, the cleavage of ...fluorescent protein Fluorescent 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...near-infrared fluorophores. Addgene's collection contains many self-labeling protein fusions as well as ...

...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...

...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...

...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 ...

...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...

...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...

...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 ...

...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...

...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...