...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...

... System Contains 336 pFusX clones allowing for Golden Gate assembly of 15-16 RVD TALEN inserts in one ...plasmids of pre-assembled trimer and tetramer RVD domains designed for rapid TALEN assembly. Kiran Musunuru...message. Both vectors utilize homodimeric FokI domains. 38143 pC-GoldyTALEN Golden Gate TALEN 2.0 36033...sequences within core promoter regions. pTAL5-BB contains the GAL1 promoter, placing TALORs built into this...under galactose-inducible expression. pTAL6-BB contains the TEF1 promoter, giving constitutive expression...mutation induction when using TALENs. The FokI domains (DD, RR) used are obligate heterodimers that require...require cloning of left and right TALEN monomer proteins into opposite vectors. 37276 pCS2TAL3-RR 48636...

...reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base... been designed for use in Drosophila and other insects. CRISPR... been designed for use in Drosophila and other insects. Cut Fully functional CRISPR/Cas enzymes will introduce...joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often...repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs...much lower efficiency than NHEJ. ID Plasmid Gene/Insert Promoter PI Publication Nick CRISPR/Cas nickase...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type...

...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...

...) that contains dCas9 fused to N 51025 pSLQ1661-sgMUC4-E3(F+E) Lentiviral vector that contains sgRNA targeting...CRISPRi is a system that can regulate a genome instead of modifying a genome. 1. Two-plasmid CRISPRi system...knockdown The first plasmid ( pdCas9_bacteria ) contains an anhydrotetracycline (aTc)-inducible dcas9 gene...resistance. The second plasmid ( pgRNA_bacteria ) contains a constitutive sgRNA expression cassette on a ...knockdown The first plasmid ( pdCas9_humanized ) contains a human codon optimized dcas9 gene under the control...promoter. The second plasmid ( pgRNA_humanized ) contains a murine U6 promoter controlled sgRNA cassette...sequences in the genome. The pgRNA_humanized also contains a CMV-puro-t2A-mCherry expression cassette, useful...

... TAG Edward Lemke Browse Strains The table below highlights bacteria strains that have been modified to...Addgene's guide to plasmids and strains useful for expanding the genetic code in bacteria and mammalian...Rinehart Lab reagents tRNA/Synthetase Pairs Bacterial Strains Codon Modified Constructs The genetic code for ...genetic code can help us understand and evolve proteins for various purposes from therapeutics to biopolymers...phosphoserine GCE. For expressing phosphoserine-containing proteins using genetic code expansion without buildup of...DE3) ΔserC serC gene knocked out; for expressing proteins containing site-specific non-hydrolyzable phosphoserine...and trpT genes). Expresses yeast TrpRS/tRNA pair instead. For directed evolution of the liberated EcTrpRS...

...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...

...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 ...

...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...

...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...

...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...

...reverse transcriptase (RT) is capable of installing targeted insertions, deletions, and all possible base-to-base...joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often...repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs...much lower efficiency than NHEJ. ID Plasmid Gene/Insert Selectable Marker PI Publication Base Edit Catalytically...the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Nick ...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type...homology-directed repair (HDR). ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Prime...

...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...