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  1. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

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    ...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...
  2. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

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    ...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...
  3. Trading Spaces - Visiting Scientist Edition

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    ...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 ...
  4. Hot Plasmids: Fall 2024

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    ...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...
  5. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

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    ...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...
  6. Luminescent Imaging with Nano-lanterns

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    ...be limited by a few caveats. Since fluorescent proteins (FP) require external light activation, you can...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene...including optogenetics, biosensors, and fusion proteins. Solving the problems of low luminescence By...generates much less signal than comparable fluorescent proteins. This weak emission hasn’t stopped some scientists...scientists from using chemiluminescent proteins for imaging, but it has precluded widespread adoption of the...eliminates the need for an excitation light source. Instead, a luciferase substrate like coelenterazine is ...Nano-lanterns and showed that these three luminescent proteins can be used together in cells. As Nano-lanterns...
  7. Viral Vectors 101: Viruses as Biological Tools

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    ...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...
  8. Antibodies 101: ChIP

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    ... 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...
  9. GPCRs: How Do They Work and How Do We Study Them?

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    ...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...
  10. Antibodies 101: Normalization and Loading Controls for Western Blots

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    ...S-shaped curve. Sparse proteins may not be detected at all, while overloaded proteins may saturate the detector...choose. Housekeeping proteins as loading controls Ubiquitously expressed proteins like β-actin or GAPDH... be careful relying on so called “housekeeping proteins.” Fully quantifying a western blot requires a ...semi-quantitative western blots. No pipetter is perfect, and proteins may not transfer precisely evenly across the entire...transfer errors — not to any factors from before the proteins even hit the gel. But what if your experiment ... this S-shaped relationship poses problems for proteins of very different abundance, consider the following...controls. These are often called “housekeeping proteins” and are detected with an antibodies, just like...
  11. How to Negotiate a Successful Lab Start Up Budget

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    ...why at Experimental Designs Consulting (EDC), we instead have our clients focus on their experimental needs...parties are clear on the goals and the resources. Instead of creating a start up budget, create a start up... a start up award by giving you step by step instructions on creating a budget, making decisions based...specific aims. This is actually why a department/institute is interested in you. Your science will define...greater than $5,000 but this can depend on the institution/funding source. Major equipment includes large...academic job titles in the regions where the host institute is located. For example, salaries in Kansas versus...is important to ensure that you’re looking at institutes that have the means to support your work. For...
  12. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

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    ...cells. The Brainbow-1.0 construct contains three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP...injection to insert these Brainbow constructs into mice. When they imaged the mouse brains, they found ...Brainbow-1.1 scales up the system to four fluorescent proteins, with recombination events specified by three ...colors (n-GFP, RFP, YFP or M-CFP.) The construct contains two tandem invertible segments, each with two ...The remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system,...intensity, caused partially by fluorophore photoinstability, as well as the tendency of fluorophores to... First, they screened a variety of fluorescent proteins to find those with ideal characteristics (low ...
  13. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

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    ...the gene/insert by aligning to NCBI entry or using BLAST.   Confirming tags and fusion proteins. We’ll...of the plasmid.   Confirming the insert We usually confirm the insert through BLAST or by direct alignment...Confirming tags and fusion proteins Finally, we confirm promoters, tags, fusion proteins, and selectable markers...and user friendly, to own the process entirely remains a large undertaking. Priority number one was to...by the depositor. Depositing labs often provide insert sequences or annotated Genbank files that are useful...We look for point mutations, truncations, and insertions that could compromise function. When we do find...promoter, and rabbit beta-globin splice acceptor, contains sequence that is over 80% GC. Some IRES sequences...
  14. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

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    ... 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific... a lethal DSB. Figure 1: CRISPR selects against bacteria where no recombineering took place. ...anhydrotetracycline (aTet), respectively. pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting... point mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is.../pEcgRNA, allows it to be used in more E. coli strains including the popular BL21(DE3) (Li et al., 2021...tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of... both systems, custom spacers/gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid...
  15. Viral Vectors 101: Optogenetic Tools

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    .... Inhibitory opsins are usually light-driven pumps that pass ions (Cl-, H+, Na+) against the concentration...Light-gated ion channels Light activated ion channels (rhodopsins) either excite or inhibit neuronal activity upon... of light (Vierock et al., 2021). Step-function opsins are chimeric ion channels that turn on with a light-pulse...are many different opsin (natural and engineered proteins responsive to light in combination with retinal...activation inhibitory (ArCH) proton pump passing ions against the concentration gradient. Created with BioRender.com...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate...partners such as light, oxygen, and voltage (LOV) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2...
  16. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

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    ... the overhangs of the assembled inserts. After ligation of the insert into the vector, the BsaI site is...transformants by colony PCR confirmed the insertion of the Lambda insert at the same high frequencies as the...single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic pathway... BsaI, and a DNA ligase such as T4 DNA ligase. Inserts, either precloned or in amplicon form (shown above...ordered, pre-determined manner. If the original insert religates into its original vector, it will subsequently... assembly: High efficiency and accurate single-insert assembly Intermediate 5- or 12-fragment assembly...assembly More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...
  17. Anatomy of a Plasmid Page at Addgene

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    ...depositing lab page contains all the plasmids from the same lab while the publication page contains all the plasmids...s plasmid number here. Gene/insert: This describes what has been inserted into the backbone, and provides...the gene/insert, this information can be found here. There may be more than one gene inserted into a backbone...associated, such as when a plasmid contains certain fluorescent proteins. Finally, for many plasmids you ... backbone, and each is given its own gene/insert section on the plasmid page. Tags are listed according...or C terminus of the transcribed and translated insert. If it is an empty vector with a tag, it’s terminus.... coli lab strain DH5a, but others may require strains such as ccdB survival (for Gateway plasmids).  ...
  18. Zebrafish as a Model for Behavior: Swimming into the Optogenetic Spotlight

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    ...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers...optogenetics.  Common opsins used in zebrafish Optogenetics functions through opsins which are light sensitive...sensitive proteins. Opsins used in optogenetic studies are not native to vertebrates and are commonly introduced...fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks of life...system for behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...
  19. FastCloning: A Newer, Simpler Cloning Technique

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    ... primers used to amplify the insert have sequences specific to the insert with an additional 15-17 bases...and insert. Once you’ve confirmed that the PCR was successful, mix together the vector and insert PCR ...Connecticut. Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without... of specific sequences for base pair overhangs, insert and vector purification, and, most importantly,...independent, it does not require purification of insert or vector products, nor does it require the use...In FastCloning, you PCR both the vector and the insert in separate reactions and then combine the products...procedure To start the FastCloning process, the insert and vector are first PCR amplified. The forward...
  20. Viral Vectors 101: The Retroviral Lifecycle

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    ...well — namely that they can cause cancer via insertional mutagenesis (which HIV does not do). Furthermore... yet retroviruses are RNA-containing viruses  (Robinson et al., 1965)! You may be beginning to realize...the fact that the virus must have its structural proteins expressed from a reverse-transcribed piece of ...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... The RT protein is made by a separate set of instructions in the same producer cells as the virus. Because... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...
  21. Deaminet 2026: Breakthroughs in Base Editing, Deaminase Biology, and Therapeutic Translation

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    ...disciplines: from structural biologists resolving APOBEC proteins at atomic resolution, to cancer biologists dissecting...deaminases at atomic resolution PE6d: Processivity gains and overextension trade-offs The prime editor PE6d...transcriptase (RT) template, resulting in unintended insertions. Audrone Lapinaite (UC Irvine) presented work...explained by dNTP availability alone, indicating an intrinsic shift in enzyme processivity. Structural data ...adenine base editor ABE8e (Richter et al., 2020) remains among the most-requested base editors at Addgene... pathway for ABE8e activity. These mechanistic insights are now being incorporated into diffusion models...pathogenic SNVs, faithful detection of ABE off-targets remains challenging. Siyuan Zou (U. Chicago) presented ...
  22. Technologies Enabled by NanoLuc® Luciferase

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    ...fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins to make ... imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs) can be problematic...fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging tools but their use is limited...tissues. To avoid absorbance of heme-containing proteins in whole animals, light at or above 600 nm wavelengths...fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited by Nluc emission and the lab has...maturation time compared to similar orange fluorescent proteins. In their work, the authors noted how CyOFP1 could...of the Enhanced Nano-Lanterns to other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase...
  23. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

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    ...ancestors of Cas12 proteins. Functionally, while Cas9 contains two nuclease domains (HNH and RuvC-like...2021), which require fusing Cas proteins with additional enzymes or domains (increasing the overall size)...investigator at ICAR-Central National Rice Research Institute. When it comes to genome editing, CRISPR is a ...genome editing toolbox. However, both of these proteins are quite large — comprising approximately 1000...one to cleave both strands. Similar to how Cas proteins require a PAM (protospacer adjacent motif) near...PAM sites change for different Cas species. For instance, the TnpB from Deinococcus radiodurans (Dra2TnpB...via PCR. A 354-bp product confirms successful insertion. Alternatively, use M13R and Oligo 2 to screen...
  24. CRISPR-based Genome Editing Systems For Editing in E. coli

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    ...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...optimizing strains with low efficiency.       Sheng Yang obtained his Ph.D. from Shanghai Institute of Biochemistry...2015). In addition to the pMB1 replicon, pTargetF contains the sgRNA targeting the genomic site of interest... system.   Figure 1: The editing process begins with the addition of the sgRNA sequence in pEcgRNA...method was much simpler and took only ~32 hours instead of ~60 hours. We successfully tested the pEcCas...precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade and pCutamp...
  25. R Bodies: Membrane-Rupturing Microscopic Tools

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    ...distances. For instance, how much force can be generated by conformational changes in proteins? How can information...produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance...rupture E. coli spheroplasts to release encapsulated proteins. Video by ACS. For a more cartoony explanation...rearranging into different polymerization states. Instead, R body monomers respond to pH changes by undergoing... variety of conditions. They are composed of 4 proteins encoded in a single operon and were first expressed...
  26. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

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    ...Antibodies are composed of two light chains and two heavy chains. The ends of the branches contain the...distribute monoclonal antibodies against popular fluorescent proteins (GFP and YFP) and protein tags (...binding specificity allows scientists to target proteins of interest for labeling, quantification, purification...cell lines. The DSHB was created by the National Institute for Child Health and Human Development (NICHD)...distribution funds our operation. Antibodies are glycoproteins that are part of the humoral immune system. ...hybridoma lines, each producing a monoclonal antibody against a target antigen. This contrasts with polyclonal...popular monoclonal antibodies include those directed against human transcription factors such as PAX7, PAX6,...
  27. Custom CRISPR Screens & the Green Listed Software

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    ...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming... benefit of working with surface proteins is also that such proteins are easily studied in living cells...Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest...screen, each gene is not targeted by only one gRNA, instead many different gRNAs are used for each gene. At...molecules were involved in the phenotype. Surface proteins  My group works on immune cells in different contexts.... Thus, a screen can be used to target surface proteins to determine which contact points are involved... used to interfere with the identified surface proteins, and such antibodies could thus be interesting...
  28. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    ...pull-down assays: Epitope tagging provides a solution Proteins - the workhorses of the cell – never work alone.... It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform...complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes... mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments...for each target protein. To study 20,000 human proteins, this would be time-consuming, expensive, and ...biochemical “epitope tag” such that these tagged proteins can be immunoprecipitated using a standard tag-specific...assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed...
  29. Plasmids 101: Biotinylation

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    ...effectively biotinylate AviTag expressing proteins in bacteria, yeast, insect and mammalian cells. Check out Kay...about in vivo biotinylation of bacterial fusion proteins Biotin is an essential vitamin for life Although... immunoprecipitation experiments. Biotinylated proteins or complexes can be easily purified using columns...help with the specific elution of biotinylated proteins. For help determining which biotinylation reagent...Biotinylation Reagent Selection Tool. AviTag and BirA Proteins can also be biotinylated by exploiting the specificity...Kay et al., 2009 for methods on biotinylating proteins with BirA. You can find plasmids that contain ...Scientists have commonly used this method to attach proteins or polypeptides to a solid substrate (Holden and...
  30. Antibodies 101: Introduction to Gating in Flow Cytometry

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    ...cells, live cells, my marker stains of interest). Let’s double-click inside our gate and proceed to the...present that vary in cell type, size, and marker proteins. But even if you are analyzing a homogenous cell... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage... the magnitude of FSC, SSC, and all fluorescent stains. The cytometer's software will allow you to adjust...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...where multiple cells were clumped together. (B) Instead of FSC, one is free to use SSC for a single cell...separation pattern of positive vs. negative signal remains the same (Figure 5).   A.                                                                                                        ...
  31. Click Biology: How to Click and Play to Explore Biology

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    ...are systems like split inteins or SpyTag, which are useful for connecting proteins in the test tube or in...image, purify, or solve the structure of proteins. Split inteins are particularly useful for a kind of cellular...be similarly useful across the diverse kinds of proteins that one finds in cells. High yield of reaction...SNAP-tag and HaloTag modules, which can connect proteins to any kind of small molecule probes with different... Tag Description Humberg et al. (2025) Split inteins Cysteine-less, fast split intein; N-terminal CLm...CLm and C-terminal Aes gp41-1 Split inteins A small, fast split intein SpyTag003/SpyCatcher003 SpyTag...Tagging Kit SpyTag/SpyCatcher CRISPR-assisted insertion tagging Kit; integration of large DNA fragments...
  32. Hot Plasmids: Fall 2025

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    ...surface proteins with a membrane-impermeable biotin substrate (Figure 1). Biotinylated proteins were isolated... from Collins & Lau et al. (2024) under CC BY-NC-ND license.  In addition to chromoproteins, the ...single-cell RNA-seq data. In addition to unique proteins on each cell type, the cell-surface shared proteome...neurons (CS SPAN) featured extracellular matrix proteins, cell adhesion molecules, transporters, ion channels...been derived from just a few parent fluorescent proteins, PinkyCaMP is the first based on mScarlet. Compared...populations in vivo in both mouse and zebrafish brains. Figure 3: PinkyCaMP and FRCaMPi in use...accessibility outside of well-equipped laboratories remains limited. Members of the Stanley Qi Lab set out ...
  33. Addgene's Cancer Collection Pages Connect the Research Community

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    ...refers to 3 proteins (H-Ras, K-Ras, and N-Ras) encoded by a family of proto-oncogenes. Ras proteins are special...” shape and lack of “druggable” domains. In other words, Ras proteins naturally lack areas that are accessible...Resources National Cancer Institute: Cancer Statistics Page National Cancer Institute: Ras Pathway v2.0 Page...products. Proto-oncogenes consist of a group of genes/proteins that, when expressed, normally encourage cell ... right places. Checks and balances: protecting against cancerous cells Luckily, there are several safety...not ideal environments for many cell types. From proteins to pathways - Ras as a model When mutations do...different cancers and identifying the roles of key proteins involved, with the goal of being able to discriminately...
  34. Build Your CRISPR Vocabulary

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    ...most famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well, both natural...you. We’ll cover the common terminology for the proteins, DNA, and RNA used in CRISPR.   Figure...tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed...spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs...Cas: Different CRISPR systems involve different proteins, but many of them are called Cas, short for “CRISPR-associated...natural and engineered. What these proteins all have in common is the ability to use an RNA guide to target...Target strand: The strand of foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand...
  35. Course-Based Undergraduate Research Experiences (CUREs)

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    ... 13(4):602-606. Brian, D. 1996, Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  ...their trainees’ very way of thinking too. Albert Einstein might have put it best when he stated, “the value...metabolic pathways (precisely the type of exercise Einstein might have resented!). In 1997, Joseph Provost...structure, function, and interaction with other proteins.   It is important to note that research ...Kimball et al., 2020). Benefits and Challenges CUREs inspire students to take ownership of their work and become... him a more effective motivator (“be like Tony Robbins!” he encourages faculty mentors.)  Collaboration..., Provost partnered with colleagues at other institutions to form a support network called the MDH CUREs...
  36. RNA Interference in Plant Biology: New Tools for an Old Favorite

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    ...is fused to the DNA backbone that contains a target sequence against the fluorescent reporter in an inverted...single hairpin dsRNA is produced that contains sequence against your gene(s) of interest and the reporter... and Biotechnology from Worcester Polytechnic Institute. What is RNAi? The loss-of-function (LOF) experiment...downregulation, or “silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and... is facilitated by DNA constructs that enable insertion of ~400 bp complementary to your gene target as...whereas shRNAs are exogenously derived. Importantly, instead of the assortment of siRNAs generated from a long...outside of limited, single cell protoplast analysis. Instead, microscopic plants must be visually identified...
  37. CRISPR 101: Targeting RNA with Cas13a (C2c2)

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    ... enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains to cleave target DNA... HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a molecule results in...SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent the limitations..., and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a...form of “bait” to identify new CRISPR-associated proteins within the bacterial genome. From this analysis... DNA. The HEPN domains within Cas13a are essential for RNA cleavage, consistent with known roles for HEPN...Comparison on Common CRISPR Enzymes Name  Enzymatic Domains Guide RNA Target PAM (PFS) Cleavage Mechanism ...
  38. CRISPR/Cas9 FAQs Answered!

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    ...correct insert (gRNA) in pX330/pX335 with BbsI/AgeI double digestion. As a successful insertion will destroy...vector (like PX330). For large changes (>100 bp insertions or deletions), we typically use plasmid donor...homology arms on each side flanking your desired insertion or mutation. Each arm is around 800 bp. For transfection...CRISPR/Cas9 system to create specific mutations or insertions by Homologous Recombination (HR), what is the...purified long oligos. For large changes (>100 bp insertions or deletions), the Zhang lab typically uses a...homology arms on each side flanking your desired insertion or mutation. Each arm is around 800 bp. For transfection... What is the maximum amount of DNA that can be inserted into the genome using CRISPR/Cas for Homologous...
  39. FPbase: A new community-editable fluorescent protein database

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    ...users can submit new fluorescent proteins, add data to existing proteins when missing, or edit inaccuracies... specific protein (for instance: EGFP).  Users can “favorite” certain proteins, or create custom protein...recognized by the 2008 Nobel Prize, fluorescent proteins (FPs) have become one of the most indispensable... one might require when making such a decision remains spread across literature in publications that introduce...community resource for information about fluorescent proteins.  It was designed with the goal of encouraging...Protein Properties provides a master list of all proteins in the database, and the Interactive Fluorescent...moderate your own version of fpvis.org with the proteins of your choosing!), and they can be downloaded...
  40. K. phaffii: Rising to the Occasion

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    ...recombinant proteins a cinch. This is because it doesn’t secrete its own endogenous proteins, making the...affordable, and it grows fast to boot. The yeast strains S. cerevisiae and S. pombe have dominated the research... between two of the most frequently used yeast strains: S. pombe (fission) and S. cerevisiae (budding)...established in yeast, including a widely used Parkinson’s experimental model. In fact, approximately 25%...differences between K. phaffii and other yeast strains.     S. cerevisiae S. pombe K. phaffii...and bears more similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii...was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries as...
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