... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ... at too high a concentration.      Figure 2: Examples of different permeabilization strategies...cause some confusion. Paraformaldehyde: Many IF protocols call for paraformaldehyde (PFA) as the fixative...recover good antibody binding by tweaking those parameters, but in some cases your antibody may just not...

...from GFP and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising..., V163A EBFP F64L, S65T, Y66H, Y145F   Table 2: Functional role of specific mutations in GFP derivatives... color also exist with only slight variations separating them.  Please note that many fluorescent proteins...(FACS): This is a type of flow cytometry that separates mixtures of cells into distinct populations based... fluorescent signal. Thus, FACS can be used to separate cells expressing GFP from cells that are not. ...

...be decorated with proteins and remain functional, 2) break membranes outside of their natural context ...PubMed PMID: 3759909. PubMed Central PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies...toxicity of R-body producing recombinant E. coli on paramecia. European Journal of Protistology 48, 290–296 ...

...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann...

...go anywhere. The team should initially consist of 2-3 individuals that are aligned in vision and outcomes... the technology. This transition takes around 1 – 2 years. The product is the measure of success. Technology...constantly practicing. “The expenditure of effort in preparation and practice is directly related to success presenting...

...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8... together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If not...pre-reads, videos or exercises to be completed in preparation for the meeting or to be read or watched during...

...your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link biomolecules...quite so much. Created with biorender.com Steps 2-4 are not found in standard IHC/ICC protocols and ...to ~4-10x linearly! This expansion physically separates molecules while maintaining their relative positions...pre-expanded samples can sometimes take on a fragmented appearance post-expansion, but that doesn’t mean we should...understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; Gambarotto, ...new insights into the cytoskeleton of malaria parasites including the conservation of a conoid. PLoS Biology...10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023) Magnify is a universal molecular...

...domains retain their individual activities when separated (2), and that DBD and TAs from different systems...308. PubMed PMID: 6312261. 2. Keegan, L., Gill, G., and Ptashne, M. Separation of DNA binding from the transcription-activating...PubMed PMID: 2547163. 5. Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y.l. Split luciferase...possibility for problems with one or more of these parameters can lead to a high number of false negatives ...

... to share information about the emerging SARS-CoV-2 virus quickly and openly. By sharing results faster...Open Scientific Communication Ecosystem Fig. 2: The scientific ecosystem, enriched by open access...bioRxiv. https://doi.org/10.1101/2021.09.07.459259 Sarabipour S, Debat HJ, Emmott E, Burgess SJ, Schwessinger...

...animal is given additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer ...level of antibodies is reached - typically within 2-4 months. After the animal has the desired level of...animal. The red blood cells and the serum are separated, and the serum - which contains the antibodies...

...et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...target DNA sequences using RNA-DNA base pairing. Separately, TnsABC forms a transposase complex with the ...bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family of transposons. Insertion...Sternberg group’s initial report of VchINT, three separate plasmids and multiple promoters were used to express...Additionally, by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple...

...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids...could identify the target genes of previously uncharacterized enhancers. The popular TALEN-based LITE system...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...found that the smaller version, ABE8.x-m, showed comparable editing behavior to ABE8.x-d. Overall, ABE8s ...MS, Rinaldi C, Slaymaker IM, Yen J, Young LE, Ciaramella G (2020) Directed evolution of adenine base editors...

...of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled...didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant... that don’t even exist yet? Andrew York, Maria Ingaramo, and their team at Calico Life Sciences recently...across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV shows a wealth of potential, and while...Lazzari-Dean, J. R., Lefebvre, A. E. Y. T., York, A. G., Ingaramo, M. (2024). Magnetic control of the brightness...

...linear region to solve for total expression time. 2. Timer functionality with a single gene Although it...290.5496 (2000): 1585-1588. PubMed PMID: 11090358. 2. Tsuboi, Takashi, et al. "Age-dependent preferential...possible to make a timer system composed of two separate FPs (one that matures quickly and the other slowly...s directed mutagenesis also produced the three separate timers shown in the table: Fast-FT, Medium-FT,...varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring cellular...

...of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC...deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that activates...movement in an open field box or a circular corridor apparatus using a camera or tracking software. Locomotion...the open (risky). Taking advantage of these characteristic traits, rodents can be used to study anxiety...measuring the time spent in a particular part of the apparatus, it turned out that the IL-1 beta overexpressing...

...plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G...activation via bioluminescence resonance energy transfer 2 (BRET2). Check out our blog post on GPCR signaling...created an open source resource called PRESTO-TANGO (parallel receptorome expression and screening via transcriptional...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...science.aav4294 Ozcan, Ahsen, Krajeski, Rohan, Ioannidi, Eleonara, et al., Programmable RNA targeting with the single-protein...

... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...in the bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to ...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ...buffer. You’ll want to run long enough to get good separation in your ladder, particularly in the sizes around...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...not only believe in the separation of church and state, but also the separation of fact and fiction. Our...

... fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil... tagged with a fluorescent protein or dye. It separates the cells mechanically in a vibrating nozzle, ...Schaeffer, Marie, et al. "Dynamic imaging of T cell-parasite interactions in the brains of mice chronically...

...Joachim ended up at the University of Amsterdam Chapter 2 - Joachim’s Academic and career goals. Career advice...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...green squares, purple circles and blue triangles. 2) An antibody immobilized on a bead binds specifically...protein G, a novel IgG-binding reagent. J Immunol 133(2):969-974. Hjelm H, Hjelm K, Sjöquist J (1972) Protein...controls for an IP. When possible, perform the IP in parallel with both a sample known to express the protein...identify any non-specific interactions, perform parallel IPs with beads only and an isotype control antibody...

...diffusion test or Kirby-Bauer susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results...." London: Wellcome Trust & HM Government (2016). 2. CDC Information Pages on Antibiotics 3. WHO Antimicrobial...antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because ‘antibiotic...Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in the bacterium, and...

...from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes..., Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P...

...due to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal directs...transcriptional read-through. Chris Voigt’s lab has characterized a set of prokaryotic terminators and deposited...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...experiment, it is important to consider how many parameters you want to use in a given experiment. For very...

...the chromosome, ending replication.   Figure 2: Prokaryotic chromosomal replication. Adapted from...Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998...cooperatively to separate nearby AT-rich regions of DNA. In a test tube, the DNA helix can be separated by DnaA...ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created with BioRender.com. ...

...gene activation, with some genes experiencing around 2-fold activation levels. Although other methods have.... This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou ...once), however, SAM exhibits activation levels comparable to other popular activation methods (VPR and ...

... digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw cycles...PMID: 24003179. PubMed Central PMCID: PMC3760253. 2. Rohwer, Forest. "Global phage diversity." Cell 113.2...10634178. 8. Murnane, John P., Michael J. Yezzi, and Barbara R. Young. "Recombination events during integration...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42. doi: 10.1645/GE-2792.1...

...pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish you...goes, “failing to plan is planning to fail.” Preparation is a key step to any experiment and can help ...

...Addgene swag. Good luck and happy naming!   Fig. 2: The mascot trio!     ...our new antibody mascot, who's joining us in preparation for our upcoming affinity reagent distribution...

...Halloween is only 2 days away and here at Addgene we couldn’t be more excited! As a small, tight-knit...(old board games and movies can provide great character costumes). All of this costume planning is for...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...: 26575238. 5. Crivat, Georgeta, and Justin W. Taraska. "Imaging proteins inside cells with fluorescent...

...connectivity to other data sources used in research; (2) connectivity to other tools used in research; and...by making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research ... delve deeper and look specifically at the characteristics and capabilities an ELN needs to facilitate...

...blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected...system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of filter paper, ...range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated, the proteins... process. The sample, a mixture of protein is separated in a gel, transferred to a membrane, stained with...tertiary structures remain intact and the protein is separated through a matrix by charge. In a denaturing western...protein is denatured to its primary structure and separated by size with smaller molecules moving more quickly... we will be focusing on denaturing westerns.  Separating a protein mix by size Sodium dodecyl sulfate-...

...would label everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore for...gating on the negative population.     Figure 2: Dump gating labels all unwanted cell lineages with...conjugated to a biotin tag but not a fluorophore. In a separate step, cells are incubated with a fluorophore-conjugated...the intranuclear ones. You do not need to use a separate fix/perm cytosolic procedure in that case.   ...cell population. To detect non-surface markers, a separate intracellular labeling protocol is necessary, ...

...to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID ...cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control Rho GTPase activity. A) Schematic...cytoskeleton. They provide cells with physical structure, separate chromosomes during mitosis, support transport ... called a katana. Opto-katanin consists of two separate blue light-sensitive systems, VVDfast to control...your protein of interest and express the other separately, then use blue light to generate CRY2-CIB1 oligomers...

...melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle...algae host for reproduction. Snail parasite Leucochloridium is a parasitic worm that infects a snail’s eyestalks...Brusca and Gilligan, 1983). Grasshopper parasite The parasitic worm Spinochordodes tellinii‘s larvae makes...ve also carved pumpkins (yes, Blugene made an appearance there too) and shared fascinatingly creepy science...Tongue-eating louse Cymothoa exigua is a marine parasite that enters fish through its gills. It attaches...replacement in a marine fish (Lutjanus guttatus) by a parasitic isopod (Crustacea: Isopoda)." Copeia 1983.3 (1983...

... a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free strand...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...formation. To further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to ...K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M.,...., Slaymaker, I. M., Yen, J., Young, L. E., & Ciaramella, G. (2020). Directed evolution of adenine base...Schwaegerle, N. D., Peraro, L., Young, L., Lee, S.-J., Ciaramella, G., & Gaudelli, N. M. (2023). Improved cytosine...., Hara, K. Y., Shimatani, Z., & Kondo, A. (2016). Targeted nucleotide editing using hybrid prokaryotic...

...environmental contaminants.  Figure 2: An Addgenie works in the tissue culture hood while wearing...doi.org/10.1290/1071-2690(2002)038<0086:caeomi>2.0.co;2 Uphoff CC, Drexler HG (2011) Detecting Mycoplasma ...of the causes of contamination. If possible, a separate lab should be made available for cell culturing...keeping them in the pockets of your lab coats. Have separate footwear for the lab to avoid bringing in environmental...wide range of mycoplasma. These methods cannot guarantee complete removal of mycoplasma but, can get rid...made permanent damage to the cell culture it is irreparable. Some cells may die due to longer incubation ...Microbiol .44: 42–49  Uphoff Cc, Drexler Hg (2002) Comparative Antibiotic Eradication of Mycoplasma Infections...

...wheat germ, HeLa, and Leishmania (Gagoski, et al 2015[2]). The proteins encoded in this set range in size ...to generate Illumina sequencing libraries using a 2-step PCR-based protocol and are compatible with single-end...centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators can...size from 4 to 156 kDa, enabling the user to characterise the efficiency of their cell-free system in correlation...

...tagging." Nature methods (2015). PubMed PMID:26322837. 2. Stadler, Charlotte, et al. "Immunofluorescence and...the fusions had some negative effect on these parameters. IT5 and IT6 displayed good binding efficiency...

...Agrobacterium infection. At approximately 7 kb, pSiM24 is 2 and 6 kb smaller than commercially available pCAMBIA...duplicated enhancer domains and characterized in studies of tobacco, Arabidopsis, and corn. For both β-glucuronidase...plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has ...functions in multiple species, including tobacco and Arabidopsis. Improving Agrobacterium binary vectors Vectors...efficiently inherited in both transgenic tobacco and Arabidopsis. Finally, Sahoo et al. demonstrate that this ...

...elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you choose ..., destabilizing the molecule and favoring the separation of the two helixes. Low pH instead results in... buffer for preserving the stability of your preparation for a long time. Tris buffer controls the pH,... if the priority is to directly use your DNA preparation in any downstream application involving enzymatic...buffer is that it allows better control of the pH preparation stabilizing the plasmid, and still allowing most...at -20°C or even at -80°C, in this case your preparation can be eluted in water or in your buffer of preference...

...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...PMID: 27882265. PubMed Central PMCID: PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced ...vectors encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the...

...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...Walton et al., 2020). Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN...

...faster when using a dual guide system.   Fig. 2: Size screening for large deletion events.   ... Biol 17: https://doi.org/10.1186/s13059-016-1012-2 Find more CRISPR Resources at Addgene: Read our CRISPRGuide...visualized by running your PCR on a gel. It’s still paramount that you sequence clones to verify that the deletions...

...Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end) - Panel Alex Tucker (Ginkgo Bioworks)...their curation of citable, linkable figures. Cause #2: Hidden “negative” data We all want to be able to ... and here I summarize what I learned both in preparation for my talk and at the minisymposium. You can...address this issue directly by creating well characterised antibodies against all human extracellular proteins...stories and read papers wherein a researcher tries 5 separate experiments at once to get confirmatory data for...