...validated? This post will go over some of the general considerations you should review before using an antibody...important to know what you are aiming to achieve. In general, these are three core goals of antibody validation...different applications. Here, I want to highlight the general suggestions given by the International Working ...these pillars is that they are intentionally generalizable and not specific to certain applications or ...analysis of transcriptomic or proteomic data from several different tissues or cell types. If your antibody...the brain, but almost none in the liver (A). In general, RNA levels tend to correspond to protein levels...s important to understand if protein:protein interactions could be a factor in your analysis. Example:...

...have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many...should be able to keep cells moist and hopefully aerated with a thin layer of molecular biology-grade agarose...sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from a 10 cm dish in 15- 20...both Hepes and bicarbonate) is brought to room temperature and placed in the hood. The medium from a 10 ...is capped, swirled and tested for the correct temperature by placing on the wrist or cheek. Within less...the agarose cloning method Test your agarose temperature. Cloning cells using an agarose overlay does ...agarose onto the recipient plate at just the right temperature. If the agarose solution is too hot, the cells...

...establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae). In Vitro, 13(4), ...packaging plasmids (three packaging plasmids for 3rd generation lentivirus production). The packaging plasmids...Insect cells are a less common option for the generation of viruses, though they are a popular choice ...from the pupa ovarian tissue of fall armyworms (Spodoptera frugiperda). Much like HEK-293T cells, they are...much larger packaging capacity — up to 38 kb! Generally, insect cell production methods produce lower ...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV and...adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https://doi.org/10.1089/10430340260355347...

... molecule). Degron tags can range in size from several kDas up to ~35 kDa and are typically knocked-in...manipulate protein expression. These tags offer several advantages over the alternative systems – CRISPR...interest to the proteasome for degradation. Like several other tags described here, the dTAG uses a small... and widespread usage, a proteolysis-targeting chimera (PROTAC) was developed for HaloTag. PROTACs are...their target protein and an E3 ligase through interacting ligands. After an E3 ligase facilitates ubiquitination... leakiness associated with the system, but the general concept of the system has remained the same. An...predict precisely how a given degron tag will cooperate with your protein of interest and how effective...

...signal you can generate without disturbing the integrity of the membrane, a concern with several of the previously...we will review how these indicators work, their general applications, and why viruses are essential to ...time from signal input to readable output, which generally exceeds that of the VSD GEVIs. However, since ... to the plasma membrane due to the addition of several targeting domains. The quencher and voltage detector...previously described systems. Several adaptations of this method have been to tag, cage, uncage, and trap...your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically encoded...can be used for chemigenetics, biosensors, and generally as biological tools, our blog has you covered....

... the Cas9 nuclease in the absence of genomic alterations. These studies utilize a catalytically inactive...dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and Fujii, 2013). They ...method can characterize nucleic acid-nucleic acid interactions at a genomic locus (Fujita et al., 2017). CRISPR-mediated...Fujita et al., 2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression...when inappropriately expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for ...specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows for the recovery...Toshitsugu, et al. "Identification of physical interactions between genomic regions by enCh IP‐Seq." Genes...

...and use a standard curve and provide some general considerations for scientists planning to use them in ...for the known concentrations (standard curve), generating an equation that allows you to determine the ... triplicate. The closeness of the data points generated by replicate values provides useful information.... With the help of graphing software, you then generate a trendline for the data. The appropriate trendline...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...To calculate the %CV of the standard curve, one averages the individual %CVs for each point on the curve...assay. Try to minimize these.  Review previous literature or results from labmates to get a sense of the...

...broke down the method into six general steps and outlined the considerations to be made during each step....that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...fixation*. Organic Solvents Organic solvents, generally acetone, methanol, or ethanol, work by dehydrating...membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and leaves ...Tween-20 are examples of non-ionic detergents that interact with lipids and proteins in membranes and permeabilize...to use your specific antibody for IF, look for general IF protocols for your sample type. Starting with...cross-linking fixation; they require an additional step generally referred to as Antigen Retrieval. I chose not ...

...by the Sternberg lab derives from Vibrio cholerae (V. cholerae INTEGRATE - hereafter VchINT) and encodes...and technical considerations Making insertions in bacterial genomes with the V. cholera INTEGRATE system... using Tn5), and as knock-in tools for human therapeutics (e.g. using Sleeping Beauty). Despite their ... CRISPR-Cas, these systems represent the next generation of tools for efficient and programmable insertions...achieved with VchINT, at two different incubation temperatures. Data adapted from Vo et al., 2020. In E...-recombineering, CRISPRa/i, and base editing. Leveraging a unique marriage between distinct biological...

... and several others can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical...by Roger Tsien, who showed that the selective interaction between a bisarsenical dye ligand (e.g., FlAsH... lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These enzymes catalyze the covalent...corollary to these innovative labeling strategies, several groups—including mine—have been revisiting the ...PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve fluorophores for live-cell and...Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids from...