...and purification An affinity tag, generally a relatively small sequence of amino acids, is basically a...tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the affinity ... tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike other epitope tags –... Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used ...crystallizing it or testing its function. Small ubiquitin-related modifier (SUMO) can help with folding and stabilization...western blots, IP, co-IP, IF, flow -cytometry, but rarely used for purification as elution requires low pH...  0.8 Affinity and Purification Very small size, rarely affects function S-tag KETAAAKFERQHMDS  1.8 Solubility...

... Backbones Flag Epitope tag c-Flag pcDNA3 or FLAG-HA-pcDNA3.1 - C-terminal Flag or N-terminal FLAG-HA...pNIC-CTHF - C-terminal TEV-His6-Flag for bacterial expression pFA6a vectors - PCR-based C-terminal...Epitope tag pcDNA3.1-HA or c-Flag pcDNA3 - Mammalian expression vector with N- or C-terminal HA tag pInducer20...trp mTagBFP2-pBAD - Protein expression vector with C-terminal mTagBFP2 tag pNIC-GST-TEV-GG - Cloning of...Gileadi lab Worm unc-54, variety of worm gene promoters C. elegans vector kit - Collection of...of plasmids from the Fire Lab L4440 - RNAi in C. elegans Also see our Worm Plasmids and...purification. Just remember to remove the stop codon for C-terminal tags and omit the start codon for N-terminal...

...expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore...codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes the histidine... ATG codon from an ACG codon using a base-editor (C>T conversion) they could induce the translation of...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464...re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency...2018). TLDR: The ACE reporter from the Harris Lab relies on the restoration of a frame-shift in mCherry ...quantifies APOBEC-Cas9 efficiency in real-time, it relies on DSBs which is not ideal for monitoring single...

... Sellnow, R. C., Boye, S. L., Coberly, B., Bennett, A., Agbandje-McKenna, M., Sortwell, C. E., Hauswirth...Backer, M. W. A., Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization...Dudek, A. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Cheng, F., Qiu, J., Carette, J. E., & Vandenberghe..., J. Y., Han, J. H., Lee, H., Han, Y. E., Rah, J. C., & Park, H. (2020). Labeling dual presynaptic inputs... Porrot, F., Bohl, D., Marmé, F., Chérel, Y., Salvetti, A., Hurtrel, B., Heard, J.-M., Rivière, Y., & ...your specific experimental system, rather than relying on other systems as proxies.  Finally, the size...consider. First, the titer required is inversely related to transduction efficacy: the higher the efficiency...

...Epitope tags are short peptides introduced at the N or C terminus of a protein that are bound by antibodies...enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the most common tags, FLAG...not at all. The introduction of a tag at the N or C terminus of a protein may disrupt the biological function...a specific application, or they exist but are unreliable for a variety of reasons (Baker, Nature). Tagging...

...both methods. These approaches have relatively low sensitivity, which can be problematic if you are trying...also influence antibody binding by altering the relative concentration of potential antibody binders and...little to no signal in the liver sample, which you do (C), so your antibody is validated! Genetic Strategies...10.1080/19420862.2015.1100787  Taussig MJ, Fonseca C, Trimmer JS (2018) Antibody validation: A view from...recommendations for improving antibody use and reliability (Roncador, et al., 2015; Taussig, et al., 2018...your target then you would expect to see good correlation between the levels of target detected in both... targeting a different epitope, and look for correlation between the results. Using antibodies against...

...please sign up here.        New toolkit for N or C terminal tagging of proteins for degradation using...has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins with mAID and other ...cytidine deaminase  Target-AID-NG, which mediated a C to T conversion in the target sites adjacent to an...Nureki’s lab engineered SpCas9 (SpCas9-NG) with relaxed preferences for the third base of the PAM so that...

...requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often...plasmids with different varieties of tags, both N- and C-terminal. Each set of plasmids uses the same restriction...

...Zhi Ying Chen, Robert C. Robbins, Mark A. Kay, Michael T. Longaker, and Joseph C. Wu. "A nonviral minicircle... generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail.... The advantages of this method are that it’s relatively simple to implement and doesn’t require time-...a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs." Plos One 11, no...Overall, integrating viral vectors efficiently and reliably generate iPSCs, but they have several safety concerns...important control. Additionally, Sendai virus doesn’t rely on promoters for transcriptional regulation, so ...Check Out Our Interview with Stem Cell Researcher Darrell Kotton Four Factors that Differentiate the Stem...

... Boeckman, J. X., Manning, C. F., Nguyen, J. T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., Van...-GABA-AR β1 (white) and anti-GABA-AR β3 (green). (C) Hippocampus labeled with anti-Kv2.1 (magenta), anti-AnkyrinG... Panels C1-C3 show magnified detail of the box in C. Image adapted from Andrews et al., 2019 under CC-... J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R., Yao, L., Yaniv... colocalization factor to assess the spatial relationship between the targets. Or, it might be sufficient...

...Oh S, Tomkinson AE, Hendrickson EA, Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations.../10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott DA... Yan M-Y, Li S-S, Ding X-Y, Guo X-P, Jin Q, Sun Y-C (2020) A CRISPR-Assisted Nonhomologous End-Joining... and how these pathways work, as this could be relevant when planning your experiment. Here, we’ll discuss...repair is that the Cas9 protein doesn’t immediately release from the broken end after cleavage, which may interfere...

...to fuse the activators instead of at the protein’s C terminus. With that new design in place, Konermann...Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki...Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann ... not managed to create a system to allow us to reliably activate essentially any gene,” she says. The ...tools because obviously, unlike the mouse, we can’t rely on genetic engineering to optimize humans for CRISPRi...

...proteins are, which makes mixing and matching a relatively easy proposition, explained Michelle Baird, who...attach the gene of interest to either the N-or the C-terminus. Even if a researcher’s gene of interest ...

...be 10 µg/mL. Pro-Tip Transducing too many cells relative to the number of virus particles reduces the transduction...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...well plate and left undisturbed for 13 days. (a, b, c) Colonies formed by expansion of single cells for ...antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or removed from ...

... was reduced by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies to isolate the ...19763938. Maucksch C, Bohla A, Hoffmann F, Schleef M, Aneja MK, Elfinger M, Hartl D, Rudolph C. Transgene expression...Growing the culture at a lower temperature such as 30°C or optimizing the incubation time can help maintain...overlap with the band corresponding to the nicked or relaxed conformation of the plasmid monomer (see this article...

...optimum of 30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular...identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance... receptor (ER) ligand binding domain (LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2 #14797). These...containing FLEx technology. Read the genOway press release for more information. References 1. Atasoy, D.,...

...NR1F3, RORG, RZR-GAMMA, RZRG, TOR RAR-related orphan receptor C RXRA Plasmids RXRA, NR2B1 retinoid X receptor... GCR, GR, GRL nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR3C2 Plasmids... MR, NR3C2VIT nuclear receptor subfamily 3, group C, member 2 NR4A1 Plasmids NR4A1, GFRP1, HMR, N10, NAK...estrogen-related receptor alpha ESRRB Plasmids ESRRB, DFNB35, ERR2, ERRb, ESRL2, NR3B2 estrogen-related receptor...Engineering Guide Empty-Backbone Resource Image is released to public domain by author. Source: Wikipedia,...Plasmids ESRRG, ERR3, ERRgamma, NR3B3 estrogen-related receptor gamma HNF4A Plasmids HNF4A, RP5-1013A22.1...MGC119326, MGC119329, RZR-ALPHA, DKFZp686M2414 RAR-related orphan receptor A RORB Plasmids RORB, NR1F2, ROR-BETA...

...store them in a -80°C freezer to prevent RNA degradation. They will degrade in a -20°C freezer.   RNA extraction...DEPC. Alternatively, glassware can be baked at 180°C for at least 4 hours.   If your final RNA sample(s...As with DNA isolation, scientists commonly rely on RNA isolation kits to make their life easier. Recently...resources on the Addgene blog Read blog posts related to plasmid protocols Find protocols for purifying...

...cloning method with high throughput capability. Engler C, Kandzia R, Marillonnet S. PLoS One 2008;3(11):e3647..., Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Nucleic...2011 Apr 14. PubMed.   Golden Gate cloning. Engler C, Marillonnet S. Methods Mol Biol. 2014;1116:119-31...cloning efforts. Golden Gate cloning technology relies on Type IIS restriction enzymes, first discovered... destination vector + insert may spontaneously religate, this transient construct retains functional Type...

...at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, or a truly... Additionally, protein lysis should be done at 4 °C or on ice, whichever is more practical. If your lysing... truly lengthy incubation (30–60 minutes) at 37 °C, if you suspect aggregation in your prep. If your proteins...protein-based blockers degrade fairly quickly, even at 4 °C, so when in doubt, make up a fresh batch.  Antibodies...and time. Though the most common conditions are 4 °C overnight or room temperature for 1–2 hours, there...sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring wash is... may need to sonicate your lysates in order to release the proteins from their binding. If you’re seeing...