...expressing TH protein indicating that there is promoter activity. Additionally, some neurons express low levels... with the same titer and serotype previously used in a different brain region that the same result will...sites will be flanked by two lox sites now in the same orientation. Next, the loxp site is excised since...since it is between two lox2272 sites in the same orientation preventing the transgene from reverting. Figure...scrambled one pair of lox sites and either used the same sequence to flank a transgene (homologous pairs)...the DIO/FLEx vector. This step is essentially the same as what was used to optimize the serotype, except...

...chemogenetic tools, where modification of cellular activity outside of your experiment site or circuit can... different phenotypes of the same cell type, are intermingled in the same brain nucleus: a serotype that...tropism between animal species or even strains of the same species. What works well in a mouse may not work...doi.org/10.3390/cells12050785 Murlidharan, G., Samulski, R. J., & Asokan, A. (2014). Biology of adeno-...Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated...10.3390/pharmaceutics13050750 Murlidharan, G., Samulski, R. J., & Asokan, A. (2014). Biology of adeno-...

... hybrid vector, as measured by β-galactosidase activity, was 81% of that from a single vector, but significantly...nbt0702-697 Hirsch ML, Agbandje-McKenna M, Jude Samulski R (2010) Little Vector, Big Gene Transduction:...Bellon I, Yin C, Chavala S, Pryadkina M, Richard I, Samulski RJ (2013) Oversized AAV Transductifon Is Mediated...

...far-red genetically encoded indicators of neuronal activity. J Neurosci Methods 362, 109314 (2021). Yu, D...., and deep penetration of red-shifted light. The same NIR FPs and biosensors can be imaged at spatial ...

...RNA cleavage, and translational repression or activation: Kellermann, Stefanie J., and Andrea Rentmeister...plasmids used to generate these fusions all use the same restriction enzymes for cloning. Furthermore, some...

... metastasis as well as teaching and mentoring activities. Follow her on twitter @zcastan or on linkedln... my mentor, since she does research at UK in the same field that I do: prostate cancer. She has guided... Pilot Program 2015-2015 Scholars need strong proactive mentors who have relevant expertise and skills...

...Griesbeck, Heim, & Tsien, 1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying antibody... However, the acceptor doesn’t need to go to the same exact location as the donor. As long as some of ...targeted nearby (such as a neighboring house on the same street as the donor), then FRET measurements are...Ebright, 2001). These Cy/NTA conjugates work the same way as NTA-sepharose beads used for protein purification... binding sites are adjacent to each other on the same protein and a negative control experiment conducted...

...effects, each group finds very low to no off-target activity at the ten highest-ranking predicted off-target...slightly different methods, they each observed the same positive phenotypes in cardiac and skeletal muscle...

... design has been shown to increase T7 promoter activity. To avoid re-cutting by Cas9 after editing, make...They have successfully used guides targeting the same or complementary strands, as well as guides facing...

...protein level at the timepoint studied Off-target activities of the RNAi Correctly interpreting your data...the abundance of the epitope in the sample, relative to cross-reactive antigens – that is, an antibody shown... recognize different epitopes of the same protein, show the same pattern of labeling, this is supportive...detecting the target-of-interest in your assay and sample-of-interest. The approaches available broadly map...determining the selectivity of antibodies for other sample types and for non-surface (intracellular) antigens...approaches used will be different depending on the sample and target type. To start the validation process...the target at a robust level, similar to that in samples of interest. If you are working with cells that...

...to researchers.  TALENs In 2010, transcription activator-like effector nucleases (TALENs) were engineered...findings of the 1952 Hershey-Chase experiment. In the same year, Rosalind Franklin and her student Raymond ...

...Resources on the Addgene Blog Learn to Use Cas9 Activators Perform Genome-Wide Screens with CRISPR Generate...investigator can be relatively certain that their samples don’t have off-target mutations if they remain ...

...spatiotemporal control of transgene expression or activity can be achieved using switches, including riboswitches...function like your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb)...systems, which use two or more AAVs to transduce the same cell, can accommodate larger, modular genetic payloads...efficient AAV-PHP.eB, developed and deposited by the same lab. AAV-PHP.eB is the most commonly requested systemic...off-target organs. AAV.CAP-B22 was developed through the same process as AAV.CAP-B10 and shows high transduction...

... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...with samples, eliminating them from the procedure removes one potential source of cross-reactivity.   While...use the same secondary antibody for any ELISA protocol that uses primary antibodies of the same isotype...-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell...used to quantify the level of target antigen in a sample. While Engvall et al. originally developed the ... proteins and small molecules from a variety of sample types (Engvall 1971). Scientists value ELISAs for...using the method. ELISA basics All ELISAs follow the same overall process. First users coat a multiwell plate...

...presence of several biomarkers together in the same tissue sample predicts a certain cancer and want to look...you will need to start with a fixed sample. Depending on the sample type, this might be frozen or formalin-fixed...you image the first target, you inactivate the fluorophores in the sample, either by chemical treatment ...al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and targets, you...known to be abundant in your samples (with a primary antibody that has the same species and isotype as the...set of samples where you label only one target in each. Compare these individually-labeled samples with ...the sample is being damaged by all the handling steps, or there’s some unexpected cross-reactivity. Keep...

...than generally reactive ones. And make sure to check for any reported cross-reactivity when planning your...minimize the risk of cross-reactivity. To further limit species cross-reactivity, try to find secondary antibodies...cross-reactivity. For example, if you are using mouse IgG2a and rat IgG1 antibodies in the same application...specific secondary antibody, rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse...Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies. Only a fraction...of the antibody molecules present in a broadly reactive secondary antibody will bind to a monoclonal primary...different isotypes and subclasses, use a broadly reactive secondary antibody to increase the chance of primary...

... replicates are repeated measurements of the same sample and are included to assess variability in the... the tau western blot several times with the same samples and compare the results across runs. If the ... wild-type and knockout samples. In this test, wild-type and knockout samples are treated with the identical...present in the wild-type samples and absent in the knockout.  If knockout samples are unavailable, try substituting...control, and cross-reactivity control when multiplexing. A, wild-type and knockout samples are the gold standard...interactions between the sample and the secondary antibody. C, a label control treats the sample to the full protocol...interpretation of results. A positive control is generally a sample or group that will have a desired response. For...

...recognize the same region, or epitope, of the target protein. These antibodies are also all the same isotype...heterogeneous mixture of many antibodies that recognize the same protein. In the immune system, antibodies are produced...peptide sequence derived from this protein. At the same time, an adjuvant such as KLH or Freund’s adjuvant... without causing high background due to cross-reactivity. When to use polyclonal antibodies One advantage...clones will still recognize your protein. For the same reason, they’re also useful in experiments where...experiment, you should either order several vials of the same lot of polyclonal antibody, or select a monoclonal...your polyclonal antibody, you’ll want to use the same positive control across experiments to be aware ...

... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...because the pair belong to the same isotype and will react with the same secondary. If you were to change...the indirect method since a secondary antibody reactive to a certain isotype will bind to all members ...that group. If you use two antibodies that are the same isotype then the secondary antibody will bind to... Figure 2: Two primary antibodies with the same isotype can not be used for multiplexed indirect...parental species which can be bound by broadly reactive secondary antibodies to that parental species....

...compare different clones of the same target to see if the results are the same/similar, emphasizing the antibody...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...confirming it will produce the same results even to a different sample. Finally, as a lab we discuss the...underlying causes of this unreliability: cross-reactivity (binds to non-target proteins), variability (...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...