We narrowed to 928 results for: Mpl

... strategy is that large complexes may not permeate through your tissue samples as homogeneously as a single...processing and sample preparation can also help reduce the levels of endogenous IgGs in your samples, but know...option is from the same species as your tissue samples. The bad news: this situation is hard to avoid ...The culprits are endogenous IgGs in your tissue sample. Most common IHC protocols use two antibodies -...targets.  Pre-formed primary and secondary antibody complexes (Figure 2B) Some labs have found that you can ...with serum from the same species as your primary/sample, and then applying this mixture to your tissue ...secondary antibodies before adding them to your sample so the secondary antibodies aren’t free to bind...

...target protein? Epitope Availability The answer is simple: epitope availability. An antibody can only bind...the researcher and ones that happen naturally.  Sample Denaturing  First, let’s think about induced conformation...frequently denature the proteins as part of the sample prep. This involves using heat and/or chemicals...being used as a marker for flow cytometry, for example, can only be recognized by antibodies that bind...epitopes available if it is being selected out of a complex mix of proteins that have been extracted from a...Picking the Right Antibody  It can get even more complex if you’re looking for proteins that are interacting...help to visualize an abstract protein during your sample prep process, imagining how it starts and if any...

...benefits gained by lowering the complexity of the screen. For example, the analysis becomes easier, the...discussed below. The software could also be used as a simple tool to rapidly find one or a few gRNAs suggested... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...-defined, hypothesis driven research question. Examples of custom CRISPR screens Differentially expressed...a lasting impression on me. The slide showed a complex signaling pathway with many receptors, signaling...be very useful for drug discovery, to dissect a complex signaling pathway, and to identify what part of...potential components of a signaling pathway. For example, one could study the cellular response to a cytokine...

... exact amount of protein in your sample, you’ll compare your samples to a standard curve. This curve is...the known samples is used to calculate the concentration of protein in the experimental samples. Get more...heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable region ...optimized for specificity and sensitivity, for example by changing certain amino acids to improve its ...expression in cell lysates, whole cells, or tissue samples. Many experiments that use antibodies use a primary...are used to detect the presence of proteins from samples containing a mixture of proteins. The proteins ...single protein from a heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA...

...promoter or the coding sequence and the template or non-template strand, among other parameters. .  Stanley...bacterial CRISPR engineering One common way of accomplishing bacterial genome engineering is done with recombineering...introduced along with an oligonucleotide repair template. Through recombineering, the locus of interest...interest is modified to match the repair template, and the locus cannot be recognized by the spacer-derived ... contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each round of...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...with XbaI to insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning...

... is completely degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning...flexible in this situation as you can simply add more beads to your sample. But, you’ll need to ensure you ...plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a ...easier than others to prevent quality issues. For example, some kits reduce the risk of ethanol carry-over...you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash...Take your average column-based miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid...can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of...

...overhangs perfectly match up. They just need enough complementary ssDNA to hold the plasmid together, even if ...if there are gaps or flaps remaining. This incomplete plasmid looks like a homologous recombination intermediate...to work fixing up the “damage” and producing a complete plasmid. Besides being compatible with any vector...either by annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are ...SLIC cloning process, a gene of interest is PCR amplified to add 5′ and 3′ homology regions from the target... separate PCR products (middle branch); or by incomplete PCR, which skips the final extension step to ...single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product...

... as these immune complexes are smaller than a primary:secondaries:conjugates complex. If you are trying...Antibody-based assays are complex and antibodies may perform differently in different samples and experimental ...primary (linear) structure using heat or acid, for example in a Western blot. If you were to try and use such...all IHC experiments are the same. Differences in sample processing, fixation, antigen-retrieval, and permeabilization...secondary antibody is ideal as it will help to amplify the signal. Some applications use either direct...that a secondary should be used when signal amplification is needed.  Species  Next, check the species...and therefore each target protein. This can be accomplished by using a panel of primary antibodies conjugated...

...degradation with dTAG-13. Example experiments See our recent papers noted below for examples of dose-responses...FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation between the FKBP12F36V-fusion and the...parental cells (or cells expressing a control -- for example, an FKBP12F36V-tagged LACZ) with dTAG molecules...5 in Nabet et al., Nature Chemical Biology for examples). Monitor neo-substrates Small molecule degraders...neo-substrates varies in different contexts (for example, IZKF1 and IZKF3 are cell-type specific transcription...description of the dTAG system. The dTAG system was employed to evaluate degradation of multiple FKBP12F36V-tagged...Central PMCID: PMC5497220. The dTAG system was employed to evaluate degradation of ENL. Cell lines were...

...deletion in MYBPC3 donated skin, blood and semen samples. Ma et al. designed plasmids containing Cas9 and..., and electroporated them with an ssODN repair template in induced pluripotent stem cells made from the...targeting efficiency with only 13% of edited cells employing HDR. Ma et al. moved forward with gRNA 1 to conduct...fertilization. Since Ma et al. designed a repair template with two synonymous mutations to distinguish it...were able to distinguish between repair from the template vs repair from the maternal MYBPC3 allele. Interestingly...from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but instead from the mother...supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing through the NHEJ...

...confidence that they aren’t simply doing an experiment wrong. However, even simple procedures like pouring...someone else's new and incredibly useful technique simply by following the abbreviated methods section found...research. I’ve talked to many researchers who have complaints about the unreliability of antibodies. IPI is...sifting through paper lab notebooks). Once you’ve completed your experiment, you’ll need to analyze your results...individuals and by organizations like Addgene (a good example is our series of blog posts on the defunct genome...promote reproducible publishing practices. One example is the publication of peer reviews so we can see... more likely that we’ll find a fluke when we’re simply trying to get our experiments to work, but part...

...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit...that transcription is now OFF.   We’ll look at examples of both natural and engineered repressible promoters...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...in mammalian cells, this system has also been implemented in a variety of other organisms, such as sphingomonads...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4... developed by Lai and Lee. LexA/lexAop is a complementary system to GAL4/UAS that functions in essentially...systems above if they all behave very similarly?” The simple answer is that sometimes it is necessary or desirable...

...choosing antibodies   Complexity is a key factor Biological systems tend to be complex with a variety of ...than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would be a better... rabbit versus mouse monoclonal antibodies for example. Rabbit monoclonal antibodies have higher affinity...affinity binding, such as immunoprecipitations. Your sample species should also be considered when choosing...immunoglobulins naturally present in the tissue sample. There are species-on-species staining protocols...proteins interacting. Experiments tend to be equally complex with users trying to probe multiple targets in ... from each other. In a direct approach, that is simple - but what about when you need to use an indirect...

...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing... tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a ...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ... the weaker binding ones first to preserve your sample. Pro tip! Because some protein is lost each time...antibodies on it if you plan to reprobe! Just as your sample protein is retained better, antibodies will stick...

...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available... the absence of methionine (i.e. the plasmid complements the deletion). Find the Plasmids Here Plasmid...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...promoter controls MET/Met gene expression, complementation should only occur when galactose replaces glucose...shown that most, but not all, S. pombe Met genes complement the corresponding S. cerevisiae met deficiencies...

...biotin-containing media. FRET is another example of a protein fragmentation complementation assay that can be used to...can be part of multiple dynamic and overlapping complexes that have distinct functions. Many existing methods...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...Protein Interactions. Split-BioID is a protein complementation assay where BirA*, an E. coli biotin ligase...proteins Y and Z interact, the two BirA* fragments complement each other to form a functional BirA* enzyme....

...with wild ones. For example, Drosophila lines with compound autosomes are completely infertile when mated...organisms with Cas9 risk creating a gene drive? The simplest way to make a gene drive is to insert an endonuclease...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...But please be aware that there are many easy-to-implement confinement strategies that are robust to human...authorities) was to detail these robust and easy-to-implement confinement strategies2. Our hope was that anyone...laboratory organisms but not wild organisms. For example, an sgRNA-only drive will spread exclusively through...with that sequence2. Both methods are easy to implement and have been tested in yeast5. Ecological confinement...

... viral polymerase complex then recognizes the encapsidated minigenome as a template for replication and...functional viral polymerase complex and utilize the minigenome as a template. Thus, it is important to ...with the viral polymerase, which is formed by a complex of two proteins: the large (L) catalytic protein...-minigenome). The anti-minigenome is used as a template by the viral polymerase to synthesize additional...addition, the polymerase will use the minigenome as a template to transcribe the reporter gene (e.g. firefly ...studied independently of other viral proteins simplifying the interpretation of results. This system can...transfected into cells are also extremely important. For example, particular ratios of plasmids encoding NP and ...

...two hours were split up between several Addgene employees, who all made the daunting task feel more comfortable...to span the first three months of my six month employment. In the beginning, I spent a lot of time shadowing...everyone was very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition to...teams at the company. This provides new (and old) employees with information on how Addgene operates as a ...most meetings are scheduled to take place and employees are generally expected to be available. In addition... core hours) at a time that works for you. For example, if I needed to go to an appointment at 3 PM, I...finished on time, there were no issues. Another example of helpful flexibility was the ability to work ...

...safe to say the sample had their attention! They isolated DNA from the sample, amplified the CRISPR locus...lab often tests samples of new bacteria from anywhere they can find, whether the samples are from a rain...might be phylogenetically classified as the more complex Class I systems that contain multiple subunits,...Cas7-11 appeared to have derived from the more complex type III-A and III-B systems. Though it was composed...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ... matter – the mutated copy could be replaced by simply dragging and dropping in a functional copy of the... also compatible with both AAV and adenovirus templates. The system, which is larger than Cas9, can also...