...optimization can be performed to both maximize the separation and reduce any background staining. This can include...clone names, which can allow you to search different providers for the same clone. This can be particularly... process, one must first confirm that a candidate antibody can detect the antigen of interest in the given...proteomic and transcriptomic datasets can be used to select a candidate cell line (e.g. DepMap) – you’ll need...PFA-saponin, PFA-triton). The fix/perm solutions can make a significant difference in the efficacy of your antibody... this post, we will describe the approaches that can be used to determine selectivity of an antibody for...line approach Genetic knockout-based validation can conclusively prove the ability of an antibody to ...

...recombinase-dependent vectors, this can cause challenges for the experimental design as these vectors can exhibit spurious...studying how neurons are physically connected, we can begin to understand how the brain works. It’s kind...roll) neurons! Neurons are really cool because they can transmit information, for example, from one of the...looking at how neurons are physically connected we can begin to understand how the brain works. There are... (Fig. 2). Viruses as neuronal scalpels Viruses can be injected into the brain and efficiently deliver...finding a virus that works well for retrograde tracing can be difficult. The in vivo workhorse adeno associated...rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at...

...information so you can tap them for advice. As a future alum, these are people you can “cold call” and ask...someone you trust. A good coach can make a big difference, if you can find one. Create your own peer mentoring...if there are other ways they can help you grow. Mentoring relationships can last for an hour or for a year...First, I will describe some of the many ways you can approach finding someone to give you advice and guidance...the training or skills you want to learn. Mentors can be senior to you or peers you admire.  If you see...away We should all have a “posse” of advisors who can help us in different ways and in diverse areas. It... you present or see your work product in action) Can help you learn the local “ropes” and become an insider...

...2 nm, which can lead to higher resolution for super-resolution microscopy. Nanobodies can be labeled with... the above listed molecular techniques, but they can also be expressed in bacteria and extracted with ... to occur: A) Multiple polyclonal secondary IgGs can bind to different epitopes of a primary antibody....multiple labels (green circles in figure 2 below) can be attached to each antibody. But being big is a... target antigen and the fluorescent label, which can reduce the potential image resolution of super-resolution...recognize only one primary antibody. This process can be avoided direct antibody fluorophore conjugates...Reagent Pros Cons Antibodies Bivalent Polyclonal Can be labeled with multiple dyes Commercially available...

...), which can be fused to recombinant proteins for one-step purification with glutathione, can also protect...instability. Prokaryotes can also have a hard time folding eukaryotic proteins. You can get your bacteria to...ubiquitin-related modifier (SUMO) can help with folding and stabilization, as can maltose-binding protein (MBP...Overexpression can also lead to insolubility, and aggregated protein is not useful protein. MBP tags can help with...biologists who need to overexpress and purify proteins can face any number of technical challenges depending...antibodies available for these tags really are good and can be used for western blots, IP, and affinity purification...unlikely to affect function, His-tagged proteins can be purified using metal-affinity chromatography, ...

... of the seemingly simple actions we can take to help ourselves can be an appropriate first step. Check...You can work later (in the evening, or in a few years) when life responsibilities are less You can work... a charitable cause.  While I can’t make it “all better” for you, I can offer some tips and tactics that...-don’t list Prioritize! Some things can wait and some things can’t. I kiss my kids good night every evening.... Tell your mom you can’t make it to dinner this week. One swim lesson missed can mean a world of mental...soccer game. Tip: take turns going; that way you can have someone there clapping more often than not. ... your career will suffer. And, yes, your partner CAN take the kids to the doctor without your help. Your...

...enzymes that can convert certain chemicals into fragrances that they can smell, or enzymes that can create hydrogels...that students can easily sense, whether that’s bright fluorescent proteins that they can see, enzymes ...that they can see, smell, and touch. Along the way, we hope they’ll also see how fun biology can be! What...This is like taking an engine out of the car – you can still use the engine as a power source, but now you...replicating organism. Furthermore, cell free systems can be freeze-dried into pellets (FD-CF pellets), leaving...Most importantly, experiments for FD-CF reactions can be fun and engaging for students. In our BioBits ... hydrogels that they can touch. With BioBits, we want students to make the connection between the facts...

...acid tag (DYKDDDDK) that can be added multiple times (often 3X FLAG, but can be more!) to a protein’s ...immunofluorescence where an antibody can’t be used or that step can be skipped. It has the flexibility ...compatible, and doesn’t come with a high background can be a huge challenge. Epitope tags eliminate the need...the antibodies used to detect them, and how they can fix your antibody woes.   What are epitope tags?...possible, without the need for an antibody. Tags can enable the separation of detection of a mutant protein...when both are expressed in the same system. Tagging can also aid in protein purification! In this blog, though...s termini to increase detection. The tag can also be removed by treating the protein with enterokinase...

...neurons, you can see how it contributes to behavior or pathology, and by disabling neurons you can figure out...days. Addgene: Can you tell us a little more about this new two-color tool and what it can enable? Boyden... maybe we can get a handle on some of these mysteries. Another application is clinical; can we find ways...the engineering of its function." Once scientists can do that, they will not only understand how the brain...neuroengineering Addgene: For those who may not be experts, can you give me a brief history of optogenetics? How ... much, much, much more light-sensitive. Then you can use dim blue light and red light together. Understanding... some of the big problems that optogenetic tools can help us to address? Boyden: There are two concrete...

... is a 26 kDa tag which can be isolated with glutathione resin. Like MBP, it can be removed via protease...Some of these tags can also be used for protein purification, just as affinity tags can sometimes be repurposed...With a little thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity...used primarily as protein purification tools (and can have secondary functions as detection aids.) When... naturally exist in most expression systems that can be added to the N or C terminus of your protein of...Maltose binding protein (MBP) is a 45 kDa tag which can be purified (along with its fusion protein) with ...with amylose resin. The MBP tag, although large, can actually increase expression and solubility of the tagged...

...system. The first two can serve as controls in a variety of experiments, while rtTA can be used to activate...that can be used to deliver fluorescent proteins to cells as determined by the serotype. You can use these...that you can use to map neuronal connections. Expanding the viral vector knowledge base: You can help! We...from select vectors in the repository yourself, you can now order ready-to-use virus directly from Addgene...many researchers we’ve met with have asked if we can help them share and distribute other reagents. Virus...while still ordering viral vectors. Virus production can be a frustrating process that requires a large investment...viral production and testing so that researchers can use our educational resources and experience to their...

...factors that can affect DNA stability in solution are pH and DNase contamination.  Extreme pH can denature...sample over time and determine which experiments you can effectively use the DNA. pH and DNases affect DNA...stranded DNA are repaired by base excision repair, but can lead to mutations in single stranded DNA undergoing...undergoing replication.   DNase on the other hand can digest and degrade the double helix, fragmenting plasmid...degradation, and/or the use of chelating agents that can be introduced in the purification protocol to remove...substantial degradation and which experiments you can use your plasmid.  DNA elution choices: TE, Tris ...the stability of your DNA, it will limit what you can do with these samples. This is because divalent cations...

...tissue sample can also need significant optimization for the sample type and size.) It can be quite the...protein and DNA are no longer bound, and the DNA can then be isolated and studied through a variety of...application for exploring these interactions, and can give valuable insight into DNA expression and protein...approach for the faint of heart!  The Process ChIP can be done for either tissues or cells. The process ...most ChIP experiences, but the increased binding can mask the epitopes antibodies recognize. It is therefore...before moving on to the next step. Typically, this can be done by cutting the tissue sample up and then ...usually the preferred method, enzymatic digestion can be appropriate when your protein of interest is a...

... gradients can be informative, so spend some time thinking about which type of gradient can help answer... gels can overheat. Running your transfer in the cold room or packing the box with ice packs can help ...from the same lot as you need or can get from the manufacturer. You can also consider using monoclonal ... high, you can consider adding a detergent like Tween-20 to your washes — although this can be harsh and...Western blots can be tricky to get right. If you’ve thoughtfully done the technical design of your blot... optimization and troubleshooting.   Before you can optimize or troubleshoot a western, you’ll need to...Created with biorender.com   Alternatively, you can run a reagent gradient. In this approach, you’ll ...

...is stored in the cloud, so you can access it from anywhere, and it can be shared with others. The Web ...Connector, papers can be saved from Firefox or Chrome browsers with just one click. Papers can be cited in ...LibreOffice, or Google Docs. Files can be synced across devices, and you can share your library with others...calendar after completing your writing goal, which can serve as positive reinforcement for a habit. The ...technique timer  One time management technique that you can use as part of your writing schedule is the Pomodoro...is called a “Pomodoro.” After four Pomodoros, you can take a longer 15-30 minute break. The method is named...word suggestions. De-jargonizer The De-jargonizer can identify jargon in your writing. While it’s not possible...

...inactivating genes. CRISPR can target a genomic sequence and create a break that can be repaired by homologous...designed in the lab. Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila...This final product of non-homologous recombination can survive positive selection, but it will be sensitive...selection marker. Since an antibiotic resistance gene can affect a cell's phenotype, researchers usually remove...through the insertion of flanking LoxP sequences, you can remove the gene through the addition of Cre recombinase...the neomycin resistance gene is floxed so that it can be removed post-selection through Cre-mediated recombination... (knocked in). As seen in the above example, you can remove a floxed resistance gene using Cre recombinase...

... equipment can be defined as equipment with capital costs greater than $5,000 but this can depend on the...where you can start to identify which items aren’t critical to your research success. If you can get away...loss of time, can exceed the cost of the new equipment. That’s why a resource budget can help you compare...your research, negotiations can be much easier. However, negotiations can still go horribly awry when ...resources needed to accomplish your science, and can include: equipment, reagents, animals, glassware,...emotional weight. Therefore, too much focus on money can dissuade a productive negotiation. A more productive...much those resources will cost and how these costs can be reduced. Negotiating is easier when both parties...

...targets by CRISPR can accelerate the drug pipeline. High-throughput CRISPR screens can be used to inhibit... may be most effective. Once drug candidates are isolated, they can be validated for specificity to their...or their direct target can be identified if it isn’t known. If a strong candidate emerges, downstream optimization...added QC is where CRISPR can save time and money to make sure quality candidates are selected.      ... targeted design. Alternatively, a disease model can be generated, and drugs screened for a change in ... workflow? CRISPR both speeds up the process and can help weed out a lot of poor drugs before time and... affect disease progression or onset. These hits can reveal individual genes or sometimes even genetic...

...Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts in your experiments....Medicine, to learn how problematic oligomerization can be and how to avoid it in your experiments. Better...proteins are incredibly useful, chemical fluorophores can still be used in a wide variety of experiments. Read...produce reporter proteins. These toehold switches can be used to quickly detect the presence of Zika virus...School of Medicine, this post describes a tool you can use to study basic virus biology while leaving virulent...transcription as well as reporter genes. To learn how you can use minigenomes in your research, check out this ...-bodies, “force generating protein machines that can extend and retract over many microns in response ...

...system or cell type can completely fail in a different system (Issa et al., 2023). This can be particularly..., which can lead to cells in far away brain regions expressing your gene of interest. This can cause issues...circuit can occur. Promoters The promoter controls how the gene of interest will be expressed; it can be ubiquitous... chicken-beta-actin or CAG can drive strong early gene expression, but can become methylated over time...tyrosine-hydroxylase (TH), can limit which cells express your gene. Off-target expression can still occur (Fig....Using AAV vectors in complex biological systems can be tricky at best, and downright infuriating at worst... validation first.  AAV-mediated gene expression can differ extensively depending on the experimental ...