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...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...

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

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

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

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

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

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

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