...of interest and the metal ions.      Fig. 1: A sample containing a polyhistidine tagged protein...proteins within a system. This can be pretty complicated given that a single cell has tens of thousands...single protein? Oftentimes, they rely on protein purification. In this strategy a single protein can be isolated...this blog will focus on one of the most common, purification based on the polyhistidine tag. What is the ...purity (Janknecht 1991, Hochuli 1988). In this purification approach, the metal ions are immobilized on ... incorporation of a cleavage site and a post-purification cleavage step to remove the tag in order to ...2000).  One downside of polyhistidine tag-based purification, however, is the propensity for nonspecific ...

...for RaPID is an RNA motif of interest. See figure 1 below for details on the key components of RaPID. ...methods Not in a native context. While native purification preserves RNA structure and naturally occurring...used the Contamination Repository for Affinity Purification (CRAPome), to identify highly biotinylated background...research groups that performed BirA*-based affinity purification mass spec experiments. These background proteins...subtracted from Ramanathan et al.’s RaPID datasets. Applications of RaPID RaPID sounds like a nifty technique...help answer? Ramanathan et al. outline three applications of RaPID: Discovery. To discover novel RNA-... that QKI is a host protein critical to Zika replication. Human genetic disease. RaPID can also be used...

... trafficking of these biosensors.   Figure 1: Schematic of iGluSnFR3 (left). GluSnFR3 in pMinDisplay... Haoyi Qiu He Tian Every two years, we seek applications for awards to early career scientists using ...virtual conference attendance. We also opened up applications to research technicians and undergraduate researchers...mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of 3-mercaptopyruvate and generates...the chromophore that are susceptible to redox modification, the MPST hydrosulfide can be transferred to... and CNO silencing. (D) Immunohistochemistry verification of the dual virus expression in CR neurons of...where she engineers genetically encoded voltage indicators. Tian developed a video-based high-throughput...

... an entirely new affinity reagent.    Fig. 1: A comparison of an antibody (left) to several alternative...re used in a number of research and clinical applications. Most of the time, when someone speaks about...deeper penetration into a tissue, useful for applications like IHC, or be able to access an epitope folded...that would size-exclude an antibody. In imaging applications, smaller affinity reagents means higher resolution...antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in lab in situations...Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are ~60 kDa proteins...antibodies are still an excellent option for many applications, and frankly, if ain't broke, don't fix it!  ...

...see how you use them in your research!  References 1.Tang, Jonathan C.Y., et al. “A nanobody-based system...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation...

...many of which are available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity...experiments. Recombinant AAVs (rAAVs) The primary application of AAV is delivery of genetic cargo to a host...numerical designations, separated by a slash, that indicate the ITR and serotype, respectively. For example...transduced gene is episomal and does not divide and replicate in the same way the host DNA does. Because of ... since you don’t have to wait for the host to replicate the DNA before expression. Now that you’ve got... S., et al. “Various AAV Serotypes and Their Applications in Gene Therapy: An Overview.” Cells, 12(5),...

...antibody for a…less sensitive moment.   Fig 1: Blugene fading into darkness because they accidentally...compensation.    Working with antibodies can get a bit complicated, but hopefully this helps you successfully select...

...internalization of two different receptors- angiotensin II type 1 receptor and the β2 adrenoceptor. GPCRs in chemogenetics...constructed plasmids that can be used in peroxidase-catalyzed proximity labeling to quantifiably measure GPCR...In this technique, the receptor fused to APEX2 catalyzes the biotinylation of proteins near the GPCR. These...Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

..., there are two main decisions to make: 1. Which modification? and 2. Which viral vector should be used...excitation or inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression...recombinase on transgenic flox animals to introduce modifications at the DNA level in a temporally and locally... brain area, avoiding side effects from such modification in other areas. An alternative strategy is to...

...best tips beyond the basics to get you started. 1) Be visible and open to connections This is a site... example, I will often see a whole bunch of notifications “a postdoc from this Uni viewed your profile...look straight at the camera. Pro tip: Turn off notifications to your network of profile changes. If you are...

... Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo RNA...AsCas12f1. They found that this system, without any modifications, is effective in genome editing in both bacteria...therapeutically relevant gene targets and expand the application to the possible treatment of genetic disease....

...Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out the hashtag #365papers...looking for a particular protocol or are trying to replicate an experiment, the methods section probably won...is science’s way of validating work prior to publication, it doesn’t mean that there are no mistakes or...

... to the use of multiple antibodies. Figure 1: Overview of using Sandwich ELISA to quantitatively...the most common type of competitive ELISA is a modification of the sandwich ELISA as described here, the...ELISA, the only difference being the additional amplification step, which helps to visualize low-abundance...

...conclusions.  Figure 1. Potential points where DNA errors can be introduced, indicated by exclamation marks... utilize an array of tools that rely on DNA amplification and manipulation to produce and edit plasmids...places where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender....through the QC pipeline to resolve any issues.  Catching and addressing QC issues Addgene’s QC team has...has developed a process for identifying, categorizing, and resolving a variety of QC issues. This process...discrepancies can be caught prior to distribution. Catching these errors prevents future experimental issues...during analysis. These include topics such as: locations in a plasmid that are less likely to affect function...

...supervisor, even if he or she is a difficult person: 1) Understand your supervisor’s expectations Every ... series.  “The only healthy communication style is assertive communication.” - Jim Rohn, Author, Entrepreneur...I heard about supervisors in all of the above categories. In addition, I also learned about two other ...Assertiveness? An assertive person is able to communicate their ideas confidently while also considering...of the most frequent sources of conflict is miscommunication. For example, you might misunderstand your...the wrong direction. The best way to avoid miscommunication is to follow up after every meeting with an...workplace, these strategies will help you to communicate with confidence earn respect from your supervisor...

...basis of many molecular biology tools! Figure 1: Common functions of natural plasmids. Created with...Shareck et al., 2004). Plasmid categorization is complex Plasmid classification has shifted over the years...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...genes between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...poisoning, the difference is virulence plasmids! This category includes genes that help bacteria invade hosts...years, and new classifications are still being created as whole-genome sequencing becomes more relevant (... common plasmid features, such as origins of replication and promoters, check out our other Plasmids 101...

...treatment and improving patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma transposon...Mechanisms of epigenetic modifications The main mechanism of epigenetic modification is chromatin remodeling...literally means ‘on top of the genome’). Epigenetic modifications can result in phenotypic changes without any...any change in genotype. Epigenetic modifications play a large role in turning genes ‘off’ and ‘on’, signaling...two main mechanisms, either through histone modifications, which can influence the transcription of genes...hypomethylation, on the other hand, has also been implicated in the development and progression of cancer ...enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. However none of ...

...If you already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise ...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...Cas9-gRNA complex and delivered to cells using cationic lipids. Transient expression of CRISPR components...

... – a mechanical device that could be used to bend 1/8th inch steel wire into a backbone structure of a...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...biologists have always struggled to effectively communicate the nuances of protein structure in a flat 2-...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...something tangible. Under the guidance of a trained educator, these instances of focused attention become magical... their labs. Soon after we began working with educators in our Center for BioMolecular Modeling, we spun...through flexibility It is common knowledge in the education community that All models are wrong….Some models...

...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no...Guangzhou and the Science City The conference was located in the modern, relatively new “Science City” area...and technology companies and start-ups choose to locate there. I made time to go into the city of Guangzhou...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...