... 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 ...Find RvLEAMshort here!    Abe, K. M., & Lim, C. J. (2024). Small LEA proteins as an effective air-water interface...endogenous methyltransferase. The strategy is generalizable to a variety of DNA-targeting proteins, so the...DNA-binding protein (dCas9 shown here). D3L recruits the endogenous, autoinhibited methyltransferase DNMT3A...specified by the DNA-binding protein. The ADD domain of the methyltransferase binds to H3K4me0 on D3L, releasing...for enrichment and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered...

...companion post describing protein expression strains, where we go over the basics of protein expression in E. ... from two individual isolates, the K-12 strain and the B strain. K-12 was isolated from a patient in 1920...  MG1655    Wild type K-12 strain; first published sequence of an E. coli K-12 strain. F- λ- ilvG- rfb...your gene, and may have even put a fluorescent protein downstream, separated by an IRES element. You did...general lab strains designed for subcloning or protein expression. If you were to include customized strains...gene Increases the stability of certain expressed proteins endA, endA1 Endonuclease I (nonspecific cleavage...fertility plasmid. A low copy-number plasmid, encodes proteins needed for bacterial conjugation. Genes listed...

...suitable antibody against your target protein, consider fusing a protein tag to the target and using an epitope-based... T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., Van Der List, D., Murray, K. D., Engebrecht, J....with tissues), where an antibody binds a target protein in your sample, then a fluorescently labeled secondary...then you use a microscope to see where the target protein is (localization) and how much there is (quantification...quantification). But what if you’ve got several interesting protein targets and you want to know if they co-localize...visually; for example, confirming that your target protein is expressed in the hippocampus, or counting the...works as expected by using it to label a different protein known to be abundant in your samples (with a primary...

...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs... 1372038. Randall, R. E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization... BiT fragment of luciferase is embedded in the I10 domain of the human titin protein and only exposed ...fragment of luciferase binds to the BiT Tag (also cyan) embedded in the Titin I10 protein (purple) only... produced by our partners at the Institute for Protein Innovation (IPI), part of a new collection of epitope...short, linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987)...including popular new approaches like PROTAC targeted protein degraders. But CRBN has been difficult to study...

...Raguram, A., Richter, M. F., Everette, K. A., Banskota, S., Tian, K., Tao, Y. A., Tolar, J., Osborn, M. ...Lim, J., Villiger, L., Zhou, W., Jiang, K., Garushyants, S. K., Roberts, N., Zhang, L., Vakulskas, C. ...desired edit (or edits) engages the prime editor protein. The prime editor consists of a Cas9 nickase fused...they engineered a dominant-negative mutant of the protein MLH1, a component of the MutSα–MutLα mismatch repair...strategies to evolve more effective prime editor proteins, but found that the mutations that emerged depended...stability. Using a CRISPRi screen, they identified a protein that enhances prime editing in mammalian cells:...~ ~ ~ ~ ~ ~ ✖ Fuse RNA-binding protein La to the prime editor Improve RNA stability ...

...Bewersdorf J (2020) Light microscopy of proteins in their ultrastructural context. Nat Commun 11:3850. https...Prakash K, et al. 2022). So what’s a researcher to do if they want to understand the ultrastructural relationships...chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016...samples. Reduced signal can be caused by loss of protein during digestion, or masking of epitopes during...adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al. 2019). Perhaps...Tillberg PW, Chen F, Piatkevich KD, et al (2016) Protein-retention expansion microscopy of cells and tissues...tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34:987–992. https://...

...-F, but instead employ a Cas12k protein instead of three Cas proteins (Fig. 1A, bottom), and require only...hallmark of Type I CRISPR-Cas systems, in contrast to single-protein effectors employed by Type II systems...spacer sequence specifying the target site. Four proteins (TniQ, Cas8, Cas7, Cas6) that forms the QCascade...module together with the crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon...INTEGRATE. The requirement for multiple Cas proteins to form the Cascade targeting complex is a hallmark...forms a novel co-complex with the transposition protein TniQ for targeting (Halpin-Healy et al., 2020).... only two transposase proteins (TnsB and C). While requiring fewer components for RNA-guided DNA integration...

...L L K Q A G D V E E N P G P E2A: (GSG) Q C T N Y A L L K L A G D V E S N P G P F2A: (GSG) V K Q T ...vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters ...site, thereby resulting in co-expression of two proteins from a single mRNA. IRES was originally discovered...your gene of interest along with a fluorescent protein or selectable marker, it is easiest to start with...T L N F D L L K L A G D V E S N P G P * (GSG) residues can be added to the 5' end of the peptide to...Retroviral pEF1a-IRES-Neo IRES Mammalian MSCV-IRES-Luciferase IRES Retroviral pWPI IRES Lentiviral AmCyan-P2A-mCherry...

...Förstner, T. Keck,  K. Becker, et al. (2011). Nature Medicine, 18, 166–171. Pubmed. A. Ertürk, K. Becker, N. ...whole mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories...guest bloggers, Jae Lee and Pantelis Tsoulfas. Jae K. Lee is an assistant Professor at the University of...Leischner, A. Schierloh, N. Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–...2012). Nature Protocols, 7(11), 1983–1995. Pubmed. K. Chung, J. Wallace, S.Y. Kim, S. Kalyanasundaram, ... 497, 332-337. Pubmed. R. Tomer, L. Ye, B. Hsueh, K. Deisseroth. (2014) Nat Protoc., 9, 1682-1697. Pubmed...of illumination (i.e. light sheet).  This is in contrast to confocal laser microscopy where the laser and...

...Chu AW-H, Zhou J, Lung DC, Kok K-H, To KK-W, Tsang OT-Y, Chan K-H, Yuen K-Y (2020) Comparative tropism,...Xiuyuan Ou, et al. that characterized the spike (S) protein of SARS-CoV-2 (Ou et al., 2020). The authors requested...Chen X, Chen J, Hu K, Jin Q, Wang J, Qian Z (2020) Characterization of spike glycoprotein of SARS-CoV-2 on...https://doi.org/10.1038/s41467-020-15562-9 Subbarao K, Roberts A (2006) Is there an ideal animal model for...fundamental part of the scientific research infrastructure. References Chu H, Chan JF-W, Yuen TT-T, ...

... J. P. K., Liu, M.-S., Hibshman, G. N., Dangerfield, T. L., Jung, K., McCool, R. S., Johnson, K. A., &...nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs to the NUC ...lobe—do not structurally resemble any other known proteins; they are completely unique to the CRISPR system... a single unpaired strand in the center of the protein. The repeat:anti-repeat duplex pokes out from the...RNA, confirming its sequence. This region of the protein varies between Cas9 enzymes from different species.... The stem loops bind along the outside of the protein, spanning both lobes. Stem loop 1, nestled between...shows the guide RNA stabilizing the DNA-binding protein portion of the CTD, ready to check for PAM sequencines...

... like chromoproteins, fluorescent proteins, and luminescent proteins. While fluorescent proteins and luminescence...interactions with cytosolic proteins. This step is important as non-specific binding to proteins can inhibit their...inquiries.  What is a chromoprotein and how is it different from fluorescent proteins? Before going into ...into the difference between chromoproteins and fluorescent proteins, it’s important to note that despite ...distinction by name, chromoproteins are actually a subset of the fluorescent protein family. What sets them...colors in ambient light. In contrast, the widely used green fluorescent protein (GFP) gives a bright green... history of fluorescent proteins in molecular biology research, chromoproteins have a shorter history....

...such as Rep or other initiator proteins, they do not require host proteins for replication and can maintain...elements, oris interact with various RNA polymerases and proteins from bacterial hosts. In a plasmid with...as recognition sites for replication initiation proteins. These iterons vary in their sequence and number... (ori1, ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various...has its own replication initiation factors and proteins independent of the host. Host range depends on...can replicate in different bacteria and express proteins within different host organisms. pBBR1-based plasmids...further to increase the range of hosts in which proteins can be expressed. For example, scientists can ...

... E. E., Yamada, K., Kuznetsov, I. A., Rand, E. A., Ochs, C. C., Jaber, Z., Gardner, K. H., & Chow, B. ...photoswitchable elements into many other types of proteins. Here, we’ll highlight a few recently-deposited...license. A naturally occurring microtubule-severing protein, Katanin is of course named for the samurai sword...optogenetic module to activate or recruit your protein of interest, maybe you want to inhibit it. Restricting...development and a knockout is not feasible or when the protein of interest supports a variety of processes in ...CIB1 clustering. Fuse either CRY2 or CIB1 to your protein of interest and express the other separately, then...generate CRY2-CIB1 oligomers that trap the fusion protein away from its usual location, preventing it from...

...interactions in the cell, including protein-protein, protein-DNA and protein-RNA interactions. The 1980s saw...Tachihara, K., Umezawa, Y.l. Split luciferase as an optical probe for detecting protein-protein interactions...discoveries to create a new system for screening protein-protein interactions. In later years, improvements ... including protein expression and correct folding, post-translational modification, protein degradation... expression of target proteins in heterologous systems (e.g. mammalian proteins in S. cerevisiae). ...configuration of your fusion proteins physically blocks the binding sites for the protein partner or the UAS/reporter...C-terminal fusions of these proteins. This way both ‘ends’ of your protein are screened for binding. Using...

... independently. The RNA-programmed DNA-binding protein dCas9 is expressed along a CRISPR array that contains...Priest, D. G., Endy, D., Dodd, I. B., & Shearwin, K. E. (2013). One-Step cloning and chromosomal integration... a gene, it blocks the progression of the RNA polymerase, thus strongly decreasing the expression level...

...HIV-derivative viruses. For transient knockdown of protein expression, you may transfect plasmid DNA directly...post-infection mRNA knockdown (quantitative PCR) ≥3 days Protein knockdown (western blot) ≥4 days Phenotypic assay...plate so as not to disturb the transfected cells. k. Incubate cells at 37°C, 5% CO 2 for 24 hours. Day...Vector Element U6 Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts...because polyT is a termination signal for RNA polymerase III. Note that these were the prevailing guidelines...to adjust the sequencing conditions if the DNA polymerase has difficulty reading through the secondary ...

...with permission.   Mutations in the spike protein are one of the key drivers in determining whether...the current vaccines are effective or not. This protein both elicits an immune response as the target of... S, Wilkinson E, Naidoo Y, Karim F, Ganga Y, Khan K, Bernstein M, Balazs AB, Gosnell BI, Hanekom W, Moosa....2020.12.015 Garcia-Beltran WF, Lam EC, St. Denis K, Nitido AD, Garcia ZH, Hauser BM, Feldman J, Pavlovic.... In their assay, the pseudoviruses express a luciferase reporter which is used as a readout for infection...

... a complex of two proteins: the large (L) catalytic protein and its cofactor VP35. VP30 is a transcription...viral proteins (compared to viral infection). Therefore, the roles/functions of individual proteins can ... PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales." Biology ...transcribed in the cell when the appropriate viral proteins are provided in trans (see Other Important Components... they do not encode all of the necessary viral proteins. Minigenome systems have been developed for many...genome and transcribes viral genes. Four viral proteins are required for viral genome replication and ...approach. First, this would prevent the study of the proteins independently from each other (see Uses of Minigenome...

...re-exposure (2, 3). More specifically, specialized Cas proteins snip the foreign DNA into small fragments approximately...paste them into the CRISPR sequence. Separate Cas proteins then express and process the CRISPR loci to generate...out these functions using slightly different Cas proteins and PAM sequences. A total of eight evolutionarily...us know.  References Ishino Y, Hideo, S, Makino K, Mitsuko A, Nakata A. J Bacteriol. 1987 Dec; 169 (... (2007). CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, ... bacterial genome. Cas genes, which code for polymerases, nucleases, and helicases, were also disrupted...