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We narrowed to 850 results for: Met

Showing: 221 - 240 of 850 results

  1. Antibodies 101: ChIP

    Type
    Blog Post
    ...capture method such as Protein A/G beads to collect your antibody-bound samples. With a bead method, you’...isolated and studied through a variety of downstream methods, such as qPCR or next-gen sequencing. ChIP is an...cutting. While sonication is usually the preferred method, enzymatic digestion can be appropriate when your...your fragmented, crosslinked chromatin.  Whichever method you choose, you’ll need to reserve a small aliquot...DNA is now ready for downstream analysis via any methods suitable for isolated and purified DNA, such as...  Where’s the challenge in that?  So while this method is certainly long and complex, you may be thinking... be a good idea to add "patience and an extra-methodical approach" to the top of your protocols when embarking...

  2. The Many Reasons for Irreproducible Research- and a Vaccine to Eradicate It

    Type
    Blog Post
    ...research can be difficult to replicate. Unclear methods Until someone attempts to replicate the results...may be impossible to determine precisely how the methods were implemented. We run into this problem frequently...Reproducibility Project: Cancer Biology, where reported methods may not include how many tests were conducted on...focuses more attention on the results instead of the methods. Unreported flexibility and questionable research....  If we eliminate the problems above: unclear methods, QRPs, and small samples, then when replications...make research more reproducible? Combat unclear methods with open data, materials, and code Only when you...questions are important enough to be answered and the methods are appropriate to answer those questions, the ...

  3. Antibodies 101: Conjugation

    Type
    Blog Post
    ...like fluorescence microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available...2007; Stoeckius, et al., 2017). Metal isotopes are useful for mass cytometry (Bendall, et al., 2012). And...measurement in single cells. Nat Methods 14:865–868. https://doi.org/10.1038/nmeth.4380  Additional resources...antibodies for your experiments.  Many of the methods for antibody conjugation can be quite variable ...Chattopadhyay PK (2012) A deep profiler’s guide to cytometry. Trends Immunol 33:323–332. https://doi.org/10.1016...

  4. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ... about prior methods for studying autophagy and how Rosella improves upon them. Methods for studying ...Electron microscopy is a traditional method for studying autophagy. This method relies on the identification ...with or without an autophagy inhibitor. While this method does provide a quantitative measure of autophagy...Mizushima, N., Yoshimori, T., & Levine, B. (2010). Methods in Mammalian Autophagy Research. Cell,140(3), 313... D. J., Cuervo, A. M., & Seglen, P. O. (2007). Methods for Monitoring Autophagy from Yeast to Human. Autophagy...Quantitative Pho8Δ60 Assay of Nonspecific Autophagy. Methods in Enzymology Autophagy: Lower Eukaryotes and Non-Mammalian...

  5. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...delivery is achieved through the use of invasive methods, limiting researchers to accessible, spatially ...The AAV (a) capsid, (b) cargo, and (c) delivery method determine cell type- or region-specific transgene...used for expression control. (c) Viral delivery methods include focused ultrasound (FUS), intracorporeal...expression, it may be better to choose a direct delivery method over systemic delivery. Otherwise, cell-specific...immunogenicity is a concern, using a purification method that removes, not just reduces, empty viral capsids...to use. Factors to consider include the delivery method, mouse strain, target cell type or tissue of interest.... It was engineered, via the M-CREATE selection method, to cross the BBB through interactions with LY6C1...

  6. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...the transfection method (i.e. polyethyleneimine, calcium phosphate or liposomal methods), the volume of...lentivirus can be concentrated by a variety of methods such as ultracentrifugation, centrifugation on ...polyethylene glycol (PEG) precipitation. All of these methods ultimately isolate the viral particles allowing...contamination. Finally, choice of concentration method depends of a variety of factors such as resources...applications. For example, while the PEG precipitation method is relatively inexpensive and does not require ...lentivirus; consequently, this may not be the best method when pure virus is required downstream. When assessing... PMID: 11589831. Jiang W. et. al.. An optimized method for high-titer lentivirus preparations without ...

  7. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

    Type
    Blog Post
    ...added to the wells. This reaction produces a colorimetric reaction that reflects the concentration of ... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019...added and the reaction can be visualized via a colorimetric reaction. The direct ELISA is often used when...concentration of proteins in an ELISA well is based on a colorimetric reaction. Most commonly, this is a blue reaction...

  8. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...Calcium is arguably one of the most important metal ions in biology (sodium and potassium are up there...developed calcium sensors to better understand how this metal ion is trafficked and regulated. In this blog we...experiment with a sensor that is tuned to your parameters.   Kinetics How fast is the process you would...used calcium reporters with different kinetic parameters. There are three GCaMP types based on their kinetics...available with improved kinetic and sensitivity parameters. Sensitivity and Localization The calcium concentrations...green, blue, red, and far-red. There is also a ratiometric GECO sensor (GEX), where one wavelength changes...mApple (jRGECO1a) are also available. Detection methods Many calcium reporters use a single fluorescent...

  9. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...presence of trimethoprim, cultures treated with a folA gRNA were more sensitive to trimethoprim when compared...two advantages over existing bacterial CRISPRi methods. First, its modular design allows for customization...Peters et al., 2019 with permission.   Two methods to introduce Mobile-CRISPRi by bacterial conjugation... which encodes the target for the antibiotic trimethoprim. When Enterobacter aerogenes, Klebsiella pneumoniae... the folA gene product not been a target for trimethoprim, there would be no changes in sensitivity. Pooled...bacterial cloning and expression system." Nature methods 2.6 (2005): 443. PubMed PMID: 15908923. Johnson... on the Addgene blog Learn about other CRISPR methods for bacterial genome engineering   Learn about ...

  10. The Importance of a Fun Workplace: Company Culture at Addgene

    Type
    Blog Post
    .... No, we’re not all BFFs. Yes, sometimes teams disagree. Yes, sometimes work is a bummer when you’d rather... is a higher likelihood that something will go awry. Don’t plan something that will involve a huge group...the carrot (or more popularly- cookies). 3. Do something outside of your ordinary environment Team building...organizing the fun” sounds a little neurotic, but hey- sometimes fun needs to be had in an orderly and timely fashion...Communicate Make sure people know that there is something fun happening! Addgene actually has chimes outside...of our break room. A ring of the chimes means something fun is going on and to get there fast! Spread ...

  11. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...model for reprogramming somatic cells. A common method for generating iPSCs to express the transcription...diminished KLF4 expression levels; this altered stoichiometry impacted reprogramming and global gene expression...and CaMKII and RhoA sensors. Laviv, et al. Nat Methods. 2016. PubMed PMID: 27798609 Listen to our mCyRFP1...constructs with Addgene. Bindels DS, et al. Nat. Methods. 2017. PubMed PMID: 27869816 Listen to our mScarlet...PhoCl plasmids at Addgene. Zhang W, et al. Nature Methods. 2017. PubMed PMID: 28288123 Listen to Our Podcast...depositor Yannick Doyon’s lab has developed a new method to increase CRISPR editing efficiency in mammalian...cells. Their marker-free coselection, analogous to methods previously used in C. elegans, targets the sodium...

  12. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

    Type
    Blog Post
    ...somatic cells via various methods. Some of the more common delivery methods include retrovirus [2], lentivirus...transfection [9]. Many of the plasmids used for these methods can be found on Addgene’s stem cell page. On this...page, you can also find a table with a list of methods and the species they were used in. iPSCs have now...iPSCs into the cell type of choice. This step is sometimes also referred to as “directed differentiation....original somatic cells [41]. The in vivo and in vitro methods are similar except that the vectors carrying the...defined generation of human cardiomyocytes. Nat Methods, 2014. 11(8): p. 855-60. PubMed PMID: 24930130....pluripotent stem cells into a keratinocyte lineage. Methods Mol Biol, 2014. 1195: p. 1-12. PubMed PMID: 24510784...

  13. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    ...neuronal population prior to behavioral testing. One method to determine this is to examine expression of the... help to refine and optimize your stimulation parameters as well. Inhibition of neuronal activation is... other unwanted phenomenon or it could reveal something fascinating. Behavior is based primarily on careful...watch what happens. Who knows, you may observe something that could move your project onto the exciting—and...to the target neurons, appropriate stimulation parameters, etc.) The easiest way to resolve these issues...would be willing to teach you. An essential, yet sometimes overlooked, aspect of scientific research is that...comparative analysis of microbial opsins." Nature methods 9.2 (2012): 159-172.5. Goshen I, et al. Dynamics...

  14. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite in glycolysis, ...of year approaching. We’re always looking for something to brighten our day, whether it’s memories of ...extracellular oxytocin dynamics in the brain. Nat Methods. 2022 Sep 22. pii: 10.1038/s41592-022-01597-x. ...screen to select variants based on performance metrics specifically for two-photon microscopy. The resulting...valuable tool to study cellular physiology and metabolism under various conditions. LiLac has improved ... here!   Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization...Temperature Imaging with a Genetically Encoded Nanothermometer. Nano Lett. 2022 Jul 27;22(14):5698-5707. doi...

  15. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ... multiple of three will do. A popular screening method for knockouts is Tracking of Indels by Decomposition...generating a knock-in mutation, the best screening method depends on what your desired edit is. Large knock-ins...knock-ins Are you introducing a large insertion, something 20 base pairs or bigger? Then screening by PCR...indicating targeted edits.   Interested in this method, but your edit doesn’t affect any endogenous restriction...be a marker for edited cells. As with all other methods, you’ll need to further validate the specific mutations...using sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is... sequencing trace file from the donor DNA. This method can precisely estimate editing frequency in a bulk...

  16. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ...Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth.1566. Epub 2011 Feb 6. PubMed...Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth.1567. Epub 2011 Feb 6. PubMed...Lichtman JW, Sanes JR. Nature Methods. 2013 May 5:10(6):540-7. doi: 10.1038/nmeth.2450. Epub 2013 May 5. PubMed...and the neuromuscular junction. In zebrafish, the method has become very useful in lineage tracing; “Zebrabow...homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Snippert HJ, van der...

  17. An Addgene Co-op: The Intersection of Fun, Friendship, and Work

    Type
    Blog Post
    ...able to understand what metrics would be useful and how to organize the metrics in a digestible manner....building strong connections with coworkers and doing something meaningful with my career. I can’t pinpoint...summer travel plans while eating lunch. There was something so special about her pure interest and curiosity...biweekly report of our social media analytics, something that had never been done at Addgene before. This... and trampoline all at once. I was able to do something that was challenging while helping us share our...confident in myself, my abilities, and my future - something I didn’t expect from a co-op experience.  Sarah...

  18. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    .... Nature Methods. 2018. PMID: 29735997 bioRxiv preprint posted July 4, 2017 A new method to identify...quantitative analysis using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent...PNAS 2018. PubMed PMID: 29915061.        A new method to identify proteins occupying a specific genomic...investigated protein binding at the TERT gene, but this method enables the unbiased discovery of proteins involved...biotin-labeled proteins can them be analyzed by mass spectrometry allowing a researcher to specifically identify...the C-BERST plasmids here. Gao at al. Nature Methods (2018) Pubmed PMID: 29735996.   ...

  19. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    ...described thermodynamics-based methods of serotype confirmation, this method does not require any additional... and use cases in Human Gene Therapy. Existing methods for AAV quality control Historically, AAV vector...sequencing, TA-based ligation and tagmentation-based methods for AAV genome sequencing (Tai 2019; Lecomte 2015...1972). Using qPCR and restriction enzyme based methods, we showed that the DNA extract is a heterogeneous...Leenay R, May AP, Paulk NK (2019) Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged...Genomes in Academic Settings. Human Gene Therapy Methods 30:195–205 . https://doi.org/10.1089/hgtb.2019.110...

  20. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
    ...selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background. Their new method uses a selection...Reiner DJ, Goldstein B. Nat Methods. 2013 Oct;10(10):1028-34. doi: 10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed...papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released, signaling a new era...modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue to be improved...PCR-based screening to detect mutants, but this method limits screening to a few hundred worms, making...
Showing: 221 - 240 of 850 results