.... They are made up of compensation beads and a conjugated antibody. Compensation beads are a mixture between... single color control prior to compensation (B) and after compensation (C) with APC-Cy7.   FMOs FMO...your experiment, and they are non-specific. The compensation beads can bind to any primary antibody you add...up your voltages and can also be helpful for compensation. You should have a single color control for ...They may also be used as a starting point for compensation (Fig. 1B-C). However, it’s important to note...and not experimental samples, the voltages and compensation you set from your single color controls may ... the reference plots provided by the antibody companies or check the literature. Pro tip! Early gates,...

...Delivering CRISPR components to plant cells Once you have selected the correct CRISPR components for your application...deliver these components to your target cells. Remember, efficient delivery of CRISPR components is essential...organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant... academic researchers through Addgene. CRISPR components for plant genome engineering CRISPR can be used...genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting sequence and ~75...line will result in a failed experiment. CRISPR components can be expressed stably or transiently depending...delivery method and cell type in question. CRISPR components can be delivered and expressed transiently using...

... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...and one-ring pyrimidine base. This is a far more complex chemical task, not a job for a single enzyme. ...bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...removed the uracil glycosylase inhibitor (UGI) components, which inhibit BER during cytosine-to-uracil ...developed two fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding...separate and preventing repair based on the complementary strand. This could explain why the edited U ...original G is not primarily converted back to a complementary C. Optimized base editing with eOPTI-CGBE and...

...approach to monitor the composition of spatiotemporally defined protein complexes. Nature communications...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...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA*, a promiscuous...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...another example of a protein fragmentation complementation assay that can be used to study binary protein...

... in proximity by the looping of chromatin, the complex of DNA and proteins that forms chromosomes. These...of a stable cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA...genomic location of interest  When these three components are co-expressed, the gRNA targets the dCas9 ... of the CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller.... This is cumbersome and also makes it hard to compare results across multiple targets because different...with primary cells or rare cell populations. Key components of the CAPTURE 2.0 system To improve upon the... Using this tag reduces the number of CAPTURE components that need to be delivered to cells from three...

...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...Protein extracts from cells or tissues contain a complex mix of proteins seen as green squares, purple circles...protein mix and antibody-target protein immune complexes are allowed to form. Following incubation, beads...beads are used to immobilize the antibody-protein complexes, and as in the first method, unbound proteins ... need to choose the protein type that is most compatible with the host species and isotype of their primary...developed, which can be used for most antibodies compatible with either Protein A or G.  While commonly used...

...point is there are many ways to quantify the composition of a viral vector solution, and generally these...only 20% of recombinant AAV2 particles were full, compared to 50% of wild-type AAV2 stocks (Grimm et al.,...understand what these values mean and how they can be compared.   Figure 2: Label that comes on Addgene...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...the AAV genome, caused by repeats and self-complementarity of the AAV genome and the ITRs. qPCR efficiency...individual sample, this will reduce convenience and comparability because each sample with be quantified with ...linear region of the assay. This may be due to competition for binding sites between the primers and the...

... plasmids with the same ori are incompatible because they will compete for the same machinery, creating...their copy number, ori, and incompatibility groups. Note the A -C compatibility grouping is an arbitrary ...origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all of ...origin of replication wisely and avoid plasmid incompatibility In other words, the best choice of ori depends... or not you need to consider your plasmid's compatibility with one or more other plasmids. Generally speaking...plasmids for an experiment, make sure they have "compatible" ori's. See the table below for more details....details.   Common Vectors Copy Number+ ori Incompatibility Group Control pUC ~500-700 pMB1 (derivative...

...gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits and metabolic... regulators, and lots of viral components. This kit is also compatible with the Joint Modular Cloning ...noticed that studies frequently made comparisons between CRISPR components expressed from different plasmid...extensive with a supplemental add-on kit and is compatible with E. coli and/or cell-free systems. Protein... backgrounds, MoClo can be invaluable for more complex editing and screening experiments in organisms ...nature of the Fragmid system allows for direct comparisons of CRISPR parts. The Fragmid system was used ...plasmid backbone elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery...

...effects, ABEs also compare favorably to other methods. In a head-to-head comparison with Cas9, Cas9 modified...double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end joining (NHEJ), HDR occurs... Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com...represent the fourth generation of base editors. Compared to BE3, BE4 offers a 2.3 fold decrease in C→G ...frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their... 40 new ABE8 variants (Gaudelli et al., 2020). Compared to ABE7.10, ABE8s resulted in 1.5-fold more editing...higher editing efficiency at non-NGG PAM variants compared to ABE7.10. ABE8s have an improved base editing...