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...Ig) heavy chains and two Ig light chains make up each antibody molecule. The Fc region and part of the...IgG, or IgE). In the context of the immune system, each isotype is expressed at different times of the immune...system production and many more engineered methods. Each type of antibody and production strategy has advantages...advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies are derived...recognize multiple epitopes on the protein. However, each animal will make antibodies against different protein...full antibodies would not physically be able to reach. While a full antibody is ~150 kDa, a nanobody is...similar concentration of cell lysate was added to each well. Image from Sun et al., 2010.   Enzyme-linked...

...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...leading to a positive signal. The same effect can be achieved for a fluorophore that relies on dimerization ...lack of fluorescence for readout may render this approach less sensitive in most applications. Learn About...cleavage and an RNA aptamer that bound the compound malachite green (MG) to create a non-protein based visualization...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...

...adeno-associated virus (AAV), from reaching their targets. The AAV9 serotype can breach the BBB following injection...but requires a high vector dose and is unable to reach all regions of the CNS. PHP.B, a neurotropic variant...C57BL/6J mice following IV injection but fails to reach the CNS of BALB/cJ mice. These results were surprising...C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared to AAV9...antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS; yet LY6A is an unlikely candidate. It was...transduced by PHP.B. The Deverman Lab took a broad approach and searched for genetic difference between the...phenotype in mice. They took a classic genetics approach and interbred C57BL/6J and BALB/cJ mice to map...

... express a gene of interest. While the general approach for using lentivirus, infect and select, seems...mycoplasma can be extremely hard to detect with levels reaching 108 cells per ml before the media becomes cloudy... cell lines, ELISA, fluorometry, or PCR-based approaches to detect even low levels of contamination. For...Uphoff et. al. outline a do it yourself PCR-based approach complete with freely available positive controls...benefit of the multiple harvest strategy is that each day you are supplying your producer cells with fresh...envelope. One drawback of the multiple harvest approach is that it tends to result in a higher volume ... your lentiviral prep!) and infect. While this approach is a great start to a successful infection, one...

... was weird like that,” says Caitlin. A love of teaching also runs through Caitlin’s blood. “I come from...faculty members, I helped grad students who were teaching for the first time. I walk[ed them] through their...them advice, [and helped] proctor exams.” These teaching experiences led Caitlin back to being a student... signed up for a 10 week class at Iowa called “Teaching Your Research.” This course has participants take...you know about it [your area of research], and teaches you how to design a course, how to design good ...ground up.”  Science writing and editing These teaching experiences definitely weighed in Caitlin’s post-grad...but in the end, writing won out. “I considered teaching at a liberal arts school but I’ve always enjoyed...

...that many of my old suitemates stopped talking to each other if I was not there to arbitrate for them. ...sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important to recognize...occurs when two people cannot agree or get along with each other, regardless of the situation. If left unchecked... of conflict can lead to people disagreeing with each other just for the sake of disagreeing. It can be...people or teams who normally get along well with each other disagree about a specific decision, direction...responsibilities. Knowing which aspects of customer support each team “owns” is critical for day-to-day operations...everyone walk away from a conflict happy, feeling they each got enough of what they wanted. However, a poorly...

...immunofluorescence There are a couple different approaches to be familiar with when talking about IF, direct...antibody targets the antigen of interest but is not attached to a fluorescent molecule, instead a secondary...BioRender.com   Indirect IF is a much more common approach than direct IF, so we will focus on this technique...why generating fluorophore-conjugated versions for each primary antibody could get out of hand. Instead,...can also require some additional optimization as each antibody performs differently and some antibodies.... There are multiple methods for fixing samples, each with its own pros and cons. The method you use will...immunofluorescence procedure with key considerations about each step. Image created with BioRender.com   The...

...sample of your protein of interest (POI). This approach is reliable, but it’s overkill in many cases. ...normalized to the loading control: the intensity of each POI band is divided by the intensity of the loading...Figure 2: Normalization with loading controls. Under each lane, the fraction of the POI band over the loading...detected with an antibodies, just like the POI. This approach is so common that we’ve used it for our cartoon..., 2008; Gilda & Gomes, 2013). Depending on the approach you choose, your loading control measurement will...on the same gel or membrane (depending on your approach) as your final blot. Quantifying the entire lane...targets. Convenient! Comparison of loading control approaches Cartoons are nice and all, but we’ll leave you...

... was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for...The Zhang lab generated a comprehensive library of each unique TF open reading frame (ORF), barcoded for...target cells as a pooled library, which will amplify each TF to determine how its presence influences cell...atlas of directed differentiation To understand how each human TF can contribute to differentiation, the ...embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression construct. Cells...screening mechanisms, although the unique barcodes for each TF help streamline identification. While the Zhang...and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding of...

...trafficking and their detection can suffer from photobleaching and cellular autofluorescence (Tsien, 1998)... typically occurring with a fluorescence-based approach. These challenges can include background autofluorescence...plates or media, which can obscure weak signals; photobleaching from the excitation light, causing the signal...look at several applications of this technology. In each section below, we cite the original paper but link...context-dependent activity can render traditional tagging approaches insufficient. Vascular endothelial growth factor...) and by endocytic trafficking of the receptor. Peach et al. (2021) utilized HiBiT to address these challenges...kinetics of the VEGF-A and the VEGFR2-NRP1 heteromer (Peach et al., 2021). They used HiBiT and LgBiT fusion ...

...Capturing), if you ask me.  Each of the three steps need to be optimized for each specific sample type. And...including epigenetic modifications. But it's not an approach for the faint of heart!  The Process ChIP can... your protein:DNA complexes will remain bound to each other until you are ready to separate them. Most...Most proteins are not likely to remain attached to the DNA throughout the sample processing and therefore...sample may need more or less cross-linking to firmly attach the proteins to the DNA, depending on how tightly... idea to add "patience and an extra-methodical approach" to the top of your protocols when embarking on... Rachel Leeson...

...sequences (loxp and lox2272) and can not recombine with each other. When Cre is introduced to the system it binds...optimal/minimal injection titer and volume needed to achieve adequate expression in your cells of interest.  ...injection titers and volumes based on the serotype Since each serotype also exhibits distinct patterns of spread...waiting periods and/or higher injection titers to achieve adequate expression. Conducting a time course experiment...start an experiment before adequate expression is reached you may obtain a false negative or conclude the...to minimize off-target expression while still achieving adequate expression in your cells of interest..../or injection volumes may need to be tested with each new vector or lot used.   Following this stepwise...

...knockin or knockout mice. While these techniques are each useful for introducing one modification into the...engineering should be used to separately introduce each mutation independently into different mice, and ...have a combination of the mutations engineered into each of their parents. Here’s how to cross mice, using... scheme should start with parental mice that are each homozygous for one of the two mutations that you...Genotyping involves getting a small piece of tissue from each mouse (typically via either a blood draw, tail clip...in handy here! Crossing your F1 heterozygotes to each other will generate approximately 1 in 16 mice that...gametes that contain a mix of genetic material from each of the parents. If both of the mutations that you...

...organizers and moderator should have a well-planned approach to making the event interesting and engaging. ...an organizer) should set up a 30 minute call with each panelist to get to know their style and to get their...discovering interesting stories, riffs or opinions that each panelist can contribute. The moderator should identify...information and remind them about the event as it approaches. Room set-up and logistics To make a really...formality and distance that prevents interaction with each other and the audience. Put the panel on a stage...panelists in a slight semi-circle (so they can see each other) on comfortable chairs (stools if you have... better. Have a good microphone system ready for each panelist if the room needs it (lapel mics are best...

...transfection efficiency is not known, this is easily achieved by splitting transfected cells into 3 plates at...about 200 ul of culture medium containing serum in each well. A 200 ul pipettor is set to 200 ul, wiped ...ml sterile tube (for 60 mm dishes only 1.5 ml of each are used). This solution is capped, swirled and ...additional growth medium is added to about halfway up each well. Tips for using the agarose cloning method...the cell type, we sometimes add a drop of serum to each well in the 48-well plate; rapidly growing cells...making sure we always maintain a backup replicates of each clone in case of contamination. We do not normally...assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of expression ...

...vacation (New York City or Hawaii?). So how can you approach improving this transferable skill? Negotiation...one of us wins, the other loses. Although this approach to negotiation is the most straightforward, it... a loser when negotiating, what if we instead approached it as a creative problem-solving exercise? Asking... an important plans? What is most important to each person? Someone has to be the first author but ...that the brand new grad student keeps using the machine? Let’s walk through a more specific example.... on many specific details about the individuals, each example provides a scenario where both people get...to be thoughtful about what is most important to each person. It can especially be difficult to think ...

... do I fix it? When plotting two channels against each other, you should be able to draw perpendicular ...APC control, with APC and APC-CY7 plotted against each other. Since it is a single-color control, you know... Inputting compensation values differs between machines or the program you’re using, but usually you can...compensation. APC and APC-Cy7 are both bleeding into each other, as is evident by the two diagonal populations...populations are not perfectly straight, but the center of each population is in a line with the other populations... APC and APC-Cy7, with compensation panels below each plot.  The general rules of compensation apply... and APC should not require compensation between each other. We can therefore conclude that this population...

... do not require any punctures. Intratracheal instillation Intratracheal instillation is used to deliver...invasive. Restraint: anesthesia Location: trachea Gauge (tracheal tubing): 23–20 Volume: < 0.07 ml Skill ...Spinal cord 30 up to 0.01 mL Expert Intratracheal Anesthesia Trachea 20–23 (tubing) < 0.07 mL Advanced ...Intranasal instillation is very similar to its intratracheal counterpart, as both procedures target the lungs...experimental therapies or pain relief, straight to the stomach. This technique involves administering medication...

... “the photon budget”: each sample has a limited number of fluorophores and each fluorophore can emit a... a limited number of photons before bleaching. The amount of information one can extract from a biological...irreversibly with a small substrate ligand motif attached to a fluorophore (Figure 1b). Engineered ligases...phosphopantetheinyltransferase) – These enzymes catalyze the covalent attachment of a fluorophore ligand to a peptide tag (Figure...bioorthogonal chemical reactions (i.e., click chemistry) to attach a fluorophore at the specific site of incorporation... the speed and selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and...budget gets a bit thin, small-molecule labeling approaches can provide a fresh infusion of photons for your...

...significantly from lot-to-lot, and have to be re-validated each time a new lot is purchased. Monoclonal antibodies... these mixtures are defined by the manufacturer, each rAb in a mixture can be individually validated. ‌...primary antibody with the new reporter. Finally, each new conjugate will have to be tested to ensure it...therefore cannot compare Bis and Tris gels or blots to each other. Once you know your gel type, you’ll need ...amount of optimization that can be done.  Note that each type of transfer requires different equipment. Wet... long exposure times (up to thirty minutes!) to reach the limit of detection. Film also needs to be digitized...require no consumables, produce digital images, reach their limit of detection quickly (~1 minute), and...