Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

In Category

We narrowed to 851 results for: Met

Showing: 181 - 200 of 851 results

  1. Technical Design of a Western Blot

    Type
    Blog Post
    ...Journal of Neuroscience Methods, 172(2), 250–254. https://doi.org/10.1016/j.jneumeth.2008.05.00...make when designing the experiment. What detection method? What membrane? What should you block with? It...time to two days.  Indirect or direct detection method Indirect detection is the most common way to run...source, making it easy to switch from one readout method to another or change primary antibodies as needed...proteins, instead of the more common chemiluminescent methods for detection (though the enzymes used for chemiluminescence...denaturing blot.  There are a number of lysing methods and buffers to choose from. If you are starting... tissue, you’ll likely want to use a mechanical method before your enzyme lysis. For cell cultures, enzyme...

  2. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...you can detect it through the APC-Cy7 parameter in the cytometer's software. It also means we should exclude...Introduction to Gating in Flow Cytometry Antibodies 101: Flow Cytometry Antibodies 101: Flow Compensation...When analyzing your cells using flow cytometry, you are typically measuring the presence or absence of... to give you the appropriate readout. A common method of labeling proteins is to fuse them with a fluorescent...reporters, and/or dye stains used in your flow cytometry experiment are known as your "panel". In this ...strength of the emitted photons detected by the cytometer. Brighter fluorophores have a stronger signal....from: https://emea.bd.com/panel-design/en/flow-cytometry-fluochrome-brightness-spillover Excitation...

  3. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ...study protein-protein interactions. Flow cytometry Flow cytometry is used to detect proteins on the surface...expression: The nuts and bolts. Methods 116:51–62 . https://doi.org/10.1016/j.ymeth.2017.01.009 Crivianu-Gaita...immune system production and many more engineered methods. Each type of antibody and production strategy ...recognizes the primary antibody and provides the method of detection. You’ll also want to use the appropriate...Enzyme-linked Immunosorbent Assay). Immunoprecipitation Sometimes, instead of just detecting the presence of protein...the mixture. Antibodies can do this, too! In this method, antibodies bound to microscopic beads grab the...The fluorescence signal is detected by the flow cytometer and functions as a proxy for protein expression...

  4. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    ...habitat (Ronda et al., 2019). They called their method Metagenomic Alteration of Gut microbiome by In situ Conjugation...Carlotta, et al. "Metagenomic engineering of the mammalian gut microbiome in situ." Nature methods 16.2 (2019... microbes. Let’s take a look at a few of these methods below. Engineering gut microbes in situ While...Harris Wang lab at Columbia University developed a method for genetically altering gut microbiome members... and diagnostic labs, deep sequencing and qPCR methods are too costly and time intensive to process a ...microbiome, James Collins’s lab developed a paper-based method that can be used to detect microbes and biomarkers...biomarkers on paper. At the heart of their method is the RNA toehold switch, a RNA hairpin that can be designed...

  5. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer ... in a matter of hours. A major drawback of this method, however, is that a p24 ELISA measures all p24 ... direct p24 measurement. Like a p24 ELISA, this method is relatively quick, providing results in less ... on the vector backbone. When using qPCR based methods for viral titration consider using a reverse transcriptase... Functional titer is considered a more accurate method to quantify virus as it only measures infectious...infectious viral particles. One of the si mplest methods of measuring functional titer uses FACS to count the...transduction with serial dilutions of a viral prep. This method is particularly useful when vectors carrying fluorescent...

  6. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...acetone, methanol, or ethanol, work by dehydrating and precipitating cellular components. This method of fixation...introduction to immunofluorescence (IF) - a common method for visualizing molecules of interest within a ...tissue. In that introduction, we broke down the method into six general steps and outlined the considerations...saturated being 37%). Formalin solutions often contain methanol as well, in order to slow the polymerization of...recover good antibody binding by tweaking those parameters, but in some cases your antibody may just not...experiment.   Figure 1: Choice of fixation method impacts antibody binding. Formaldehyde cross-links...hidden within the folded protein (green region). Methanol dehydrates the protein, which can disrupt protein...

  7. Antibodies 101: Antibody Engineering and Directed Evolution

    Type
    Blog Post
    ...via flow cytometry for improved binding under defined environmental conditions. These methods are especially... blots, ELISAs, immunoprecipitation, and flow cytometry. The better these tools are, the better the reproducibility...easier and quicker than others. We’ve covered some methods in previous blog posts, like developing recombinant... Figure 1: Overview of antibody engineering methods. (A) Basic structure of an antibody. (B) Examples...Antibody directed evolution Directed evolution is a method employed to rapidly change a protein towards a ...directed evolution. The nice thing about these methods is that they all follow the same general cyclical...changed to further lower fidelity, like changing the metal cofactor and the dNTP ratio. The resulting random...

  8. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium, Myxococcus...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum...Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus...plasmids may have high copy numbers and reduced metabolic load (Meyer 2009). Figure 1: Idealized ...eutrophus, Azotobacter vinelandii, Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris...plasmid vectors for gene expression in bacteria. Methods in Molecular Biology (Clifton, N.J.), 765, 327–...

  9. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...then eluted. This method is appropriate for most situations.  A slightly different method is desirable if...typically carried out in one of two ways. Both methods utilize the same basic strategy: immobilize the...target protein is eluted.     In the first method, an antibody against a target protein is immobilized...if the target is present in low amounts. In this method, the free antibody is incubated with the protein...antibody-protein complexes, and as in the first method, unbound proteins are washed away and the target...protein. One of the most common immobilization methods utilizes bacterial cell wall proteins, Protein ..., the target protein can be eluted. The elution method will vary depending on the specific target protein...

  10. K. phaffii: Rising to the Occasion

    Type
    Blog Post
    ...cells and peroxisomes are where methanol metabolism occurs in methylotrophic organisms. K. phaffii is an ...obligate aerobe. It’s also a methylotropic yeast, meaning it can utilize methanol as its sole source of carbon...media change into methanol and peroxisomal autophagy can be induced by removing methanol from the media....protein animal feed. Due to increasing prices of methanol and advancements in other biomass production techniques...Preferred carbon source Glucose Glucose Glucose, methanol, glycerol Heterothallic mating Efficient...more similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a... its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, was co-opted ...

  11. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...Several methods can be used to create mutational scanning libraries. While each of these methods works ...saturation mutagenesis. Error-prone PCR: This method amplifies the wild-type template with a “sloppy...5) the addition of MnCl2. Disadvantages of this method include favoring of certain types of mutations,...assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson assembly is that the...PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best combination of library...the phage-derived uracil labeled template, this method requires further DNA shuffling to combine pairs...tested in a functional screen of your liking. This method is similar to site-directed mutagenesis (SDM) PCR...

  12. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ... resolution than using more complex protective methods or detergents like CHAPSO, while requiring a lower...particle orientations, this could be overcome with methods such as chemical crosslinking or tilted-stage imaging... stringent for eVLPs than other viral delivery methods, allowing all the PE components to be delivered...recent publication, the Liu lab showed that this method of delivery could be used to partially correct ...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

  13. IBEX Knowledge-Base: A Data Resource for Multiplex Tissue Imaging

    Type
    Blog Post
    ...multiplexed: you can image over 65 different parameters using this method (see Movie S8 from Radtke et al., 2020...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...many conventional microscopes, amplified staining methods, and oligonucleotide-based staining. Of course,..., dye inactivation details, tissue preservation method, and detergents that they used in their experiments...Screenshot showing reagent entries with associated metadata adapted from the Affinity Reagent Working Group...Human Biomolecular Atlas Program (HuBMAP) antibody metadata standards. And the dataset is rich. Experimental... host organism, isotype, clone details, vendor, method, tissue preservation, target tissue, detergent,...

  14. What's New in CRISPR - March 2020

    Type
    Blog Post
    ...acetylation or demethylation. Casilio DNA Methylation Editing (Casilio-ME) is a methylation editing tool...for linking methylcytosine oxidation to DNA repair, which increases activation of methylation-silenced genes...human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression ...Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated CBEs ...organism for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, ...Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1...Albert Cheng’s lab that enhances RNA-guided methylcytosine editing by TET1 by co-delivering TET1 and protein...

  15. Mycoplasma Contamination: Where Does It Come From and How to Prevent It

    Type
    Blog Post
    ...streptomycin. Mycoplasma can pass through filtration methods because of its ability to change shape and the ...gold standard’ for detecting mycoplasmas. In this method, the supernatant from the cell culture is added...colonies are easily visible on the agar plates. This method is capable of detecting most of the mycoplasma ...mycoplasma species, apart from a few of them. Another method used for mycoplasma detection is using PCR. In this...either species/gene-specific or universal. This method is quick, efficient, reliable and cost-effective...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of ...and this induces a noticeable color change. This method has high sensitivity but low specificity (Degeling...

  16. Antibodies 101: Reading a Flow Plot

    Type
    Blog Post
    ...how to read a flow plot.  Dot plots Flow cytometry is a method that allows users to analyze single cells...in flow cytometers, and output is generated through measurement of events. An event is something that passes...data in the literature. Data reported from flow cytometry experiments can be a little challenging to understand... read by the machine’s sensors. (Usually this something is a cell, but it could be debris or unbound antibodies...Created with biorender.com.   The goal of flow cytometry is to use increasingly more stringent markers ...in the same order. However, in an actual flow cytometry run, all markers are read in parallel.  Let’s ....  Pro tip! The forward scatter is a proxy for diameter and volume of the cell, thus allowing the exclusion...

  17. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...the AOX1 promoter whose expression is induced by methanol. Once linearized, the delivery vector can be integrated...Zeocin resistance. Then, counter-selection with methanol medium selects for those cells that have removed... In cells where recombination has not occured, methanol induces the production of the toxic MazF leading...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...the cell grows resulting in the appearance of unmethylated sites in the chromosome. The more stable endonuclease...

  18. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ...to as “Epi” illumination. Therefore these are sometimes call Epi-fluorescence microscopes. (See left image...enhance performance. In spinning disk microscopy, a metal disk containing a number of holes rotates through...only excites fluorophores within a few hundred nanometers of the glass/water interface. Super resolution...only excites fluorophores within a few hundred nanometers of the coverslip (4). Essentially, an optical...10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, ...image thick scattering samples in their entirety, something that is not possible via traditional confocal ... Conchello JA. Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello...

  19. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ... live cells. Lam et al., Nat Methods 2014 Nov 24. doi: 10.1038/nmeth.3179. Martell et al., Nat Biotechnol...Autophagy 2013 Nov 1;9(11):1852-61. A quicker method for protein inactivation Your protein of interest...advantages of Golden Gate and Multisite Gateway cloning methods developed by the lab of Joachim Wittbrodt might...process provides improvements to classical cloning methods, particularly for complex transgenesis constructs... for certain variants. Klapoetke et al., Nat Methods 2014 Mar 11;(3):338-46. For more information ...system requires less work than using established methods and minimizes some of the technical issues associated...turn RUSH and CRUSH alleles off or on enables this method to rapidly address questions of tissue-specificity...

  20. RaPID Detection of RNA-protein Interactions

    Type
    Blog Post
    ...Sometimes it feels like DNA and protein get all the attention.There are numerous ways to detect DNA-protein...created the RNA-protein interaction detection (RaPID) method. RaPID borrows the E. coli biotin ligase BirA* ...Challenges with existing RNA-protein interaction methods Not in a native context. While native purification...that the protein of interest interacts with RNA. Methods like cross-linking immunoprecipitation (CLIP) identify...interact with an RNA of interest. Current labeling methods are not ideal for in vivo work. BirA* requires ...RaPID identifies RNA-protein interactions. Both methods rely on the promiscuous E. coli biotin ligase, ...RaPID Like other BirA*-based proximity labeling methods, RaPID cannot distinguish between proteins that...
Showing: 181 - 200 of 851 results