...homology directed repair to make precise edits or knock-ins. Editing efficiency decreases drastically once your...away from the insertion site, so HDR will be difficult if there’s no PAM near your insertion site. Base ...upstream of the PAM. Even if there’s a PAM near your insertion site, the corresponding gRNA might be undesirable...multiple PAMs should have a fitness advantage over proteins with single PAM recognition. Created with...were capable of cleavage, they tested the clones against an NNN PAM library in bacteria where cleavage leads...

... your best self to the training experience.  The ins and outs of being a trainee There is a lot of advice...technical mentorship from a ‘bench mentor’. In some instances, the bench mentor is also the scientific mentor...different, so let your mentor know what yours is! For instance, some people like to ask questions throughout ... clarification at first, but in the end everyone wins. Your mentor learns where you are at and the level...

...their controls!). This blog post will discuss the ins and outs of controls for biological experiments, ... experimental (?) groups were run on a western against a protein standard (P) to check for tau molecular...antibody specificity is to test the primary antibody against wild-type and knockout samples. In this test, wild-type... each experimental run as long as the protocol remains the same.    Figure 2: Microscopy-based ...positively charged groups with generally sticky proteins such as bovine serum albumin. Secondary antibodies...all secondary antibodies are included. If, for instance, you omitted a mouse IgG2a isotype primary antibody... cells only (for example, propidium iodide only stains dead cells). Compensation controls address the ...

..., the efficiency of Easi-CRISPR for simple knock-ins and floxed models varies from 8.5-100% of pups born...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.” Genome Biol. ...standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria....remain more difficult to make. For example, large insertions, like a fluorescent protein knock-in, suffer ...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks ...other undesired outcomes, including one loxP site insertion and one NHEJ-repaired indel, often occur. ...your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et...

...into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified...systems. Insect expression systems Insect cells can also be used to produce complex eukaryotic proteins with...Researches can visualize proteins in vivo by tagging them with fluorescent proteins to study localization...localization or purify proteins to study their structure, interactions and functions. Proteins can also be purified... to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively easily...are two types of insect expression systems; baculovirus infected and non-lytic insect cells. Baculovirus...genes integrated into the insect cell genome. Both of these types of insect expression systems can be ...

...       Insert-specific primers: Insert-specific primers are designed to anneal to an insert-specific... your insert or if the insert is in your plasmid Have to make a new primer pair for each insert Backbone-Specific...the insert, insert orientation, and the sequences of the junctions between the plasmid and insert DNA....negative clone without the insert. This type of primer pair can tell you if the insert is the correct size and...orientation of the insert. Orientation-specific primers: If you need information about insert orientation,...of the insert. One member of this type of primer pair anneals to a sequence flanking the insert and one...anneals to the insert. A simple way to create this type of primer pair is to mix-and-match insert-specific ...

...reporters as well as secretory proteins or proteins targeted to membrane sub-domains. It has also been used to... reporter mRNA, which contains the tethering sites for the fluorescent proteins and an inducible promoter...and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins that are bound...the nucleus (thanks to a NLS on these proteins). The RFP proteins, however, remain bound to the mRNA since...the Wyss Institute have extended the transactivating capabilities of CRISPR/dCas9-fusion proteins and have...engineered hook proteins fused to a streptavidin core that are able to retain reporter proteins fused to the...possible protein domains containing these properties, they generated monoclonal antibodies against them, fused...

... open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been cut by compatible... allow your insert to be placed into the backbone in the proper orientation. For instance, if you were...enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert can then ...used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification ...separate your backbone away from any inserts cut out of it and your new insert from any overhangs cut off of ...these stains see the Bitesize Bio Blog and their associated manufacturers websites. These stains require...Your Insert into Your Vector In the ligation step, you mix your purified, cut backbone and insert in a...

...fused to two proteins, here proteins Y and Z, that are known to interact. When  proteins Y and Z interact...interacting proteins; nearby proteins are biotinylated when and where the two bait proteins interact, but...interact or from proteins that are just in the proximity of the bait proteins. Instead of pinpointing specific...restored, and nearby proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed in...activity is restored, and nearby proteins are labeled with biotin. When proteins Y and Z part ways, BirA* biotinylation...the biotin labeling remains even if the interaction with the BirA* fused bait proteins ceases. Lastly, Split-BioID...provides a candidate list of proteins that are part of the bait proteins’ interactome. Further characterization...

.... The Moffat laboratory maintains a website for the TKO library that contains up-to-date information and...(3) but its portability to other bacterial strains remains seriously limited, as prior optimizations are...ligase to biotinylate proximate proteins in the cell. These candidate proteins can be enriched via biotin ...of fusion proteins, requires less biotin, and shows enhanced labeling of proximate proteins. Overall BioID2...optimization of industrial yeast strains. However, industrial yeast strains are more difficult to genetically...which “bait” proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with...of RNA-binding proteins identified by editing Identifying targets of RNA-binding proteins (RBPs) has traditionally...

... next insert. Repeat as needed for more inserts. When you are finished entering your gene/insert data,...cloning a gene/insert into it. Empty backbones often contain epitope tags or fusion proteins that are appended...within your insert (or targeted by a gRNA/shRNA) and select the species that the gene or insert sequence ...include the total size of the insert and the GenBank ID associated with your insert. Pro Tip! If the correct...of the insert (when cloned into the vector backbone). If your plasmid has more than one insert, click .... For instance, if your plasmid is designed to allow you to produce large amounts of your insert in E....it. If your plasmid has multiple inserts, enter details for each insert individually.   Figure 9: Add ...

... either side of the expected insert. If the plasmid contains your insert, you will see two bands: one ...another enzyme that cuts within the insert. If the plasmid contains the insert, you will see two bands but if...the plasmid contains the insert and no PCR product if the plasmid does not contain the insert. If you’re...but it won’t tell you whether the vector contains the gRNA insert or not. A blue-white screen, however, ...sized bands depending on whether the plasmid contains your insert of interest or not. For example, choose ...the other representing the insert. If the plasmid doesn’t contain the insert, you will just see the vector...PCR to detect your insert, either by using primers that are specific to your insert or by using primers...

...isolating proteins from a specific compartment. If your proteins are nuclear- or DNA-binding proteins, you ...step: protein degradation, incomplete lysing/insoluble proteins, and protein aggregation. Protein degradation...gel box. If overheating remains an issue, which can happen with large proteins, try lowering the voltage...transferring are smaller proteins going completely through the membrane or larger proteins not transferring completely...sizes for different proteins: try using a 0.2 µm (psi) membrane for small proteins (< 15 kDa) and a 0.45...which works better for small and medium proteins. If your proteins are compatible with electroblots/dry ...migration of smaller proteins through the membrane while increasing the ability of proteins to stick to the...

...primary antibodies conjugated to fluorescent proteins, instead of the more common chemiluminescent methods...or β-mercaptoethanol.  Denaturing proteins To denature your proteins, you'll want to use SDS and either...-life; can alter proteins due to high pH Bis-tris 6–400 kDa MES for proteins <40 kDa 180 V, ...weight proteins Long running time   It’s important to note that Bis and Tris gels run proteins in different...They work well for proteins of most sizes and are worth considering if your proteins of interest vary wildly...protein you are expecting.  Fluorescent proteins Fluorescent proteins, while more expensive, allow for multiplexing.... Protein preparation Before you can blot your proteins, you’ll need to lyse and denature them. If you...

... the Institute for Protein Innovation (IPI), a Boston-based, nonprofit protein research institute, partnered... closed states, integrins are inactive, with low affinity for ligands. When integrins interact with extracellular...the role of specific integrins in cellular processes.   Figure 1: Integrins can shape into three...recognition site for one subfamily of integrins, the RGD-binding integrins. Six of the 11 IPI antibodies contain...Targeting integrins in the lab The Ha and Springer labs recently showed RGD-binding integrins demanded ...RGD-binding integrins, individually and in combination, showing that among the three αV integrins expressed...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...

...wages, overtime tracking? What are institutions doing? Institutions are now preparing to comply with the...resource on what institutions are actually doing (using the list of 340 institutions with postdocs in .... Most institutions suggest that postdocs should be paid according to the National Institutes of Health...In 2014, 89% of institutions had a minimum salary policy: of those, 51% of institutions set their postdoctoral...should be on the lookout for what their institutions, or institutions they're thinking about applying to, ...salaries will have to double at some institutions; and that institutions which can more easily provide bridge...scientists and those in technical roles. Therefore, institutions are responsible for ensuring that either all...

...integral membrane proteins and membrane associated proteins (i.e. GPI-anchored proteins), which are more... National Institutes of Health and he is currently an Associate Professor at Albert Einstein College of...tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila ...Fluorescent Proteins Browse Our Fluorescent Protein Pages Find the mOX Fluorescent Proteins at Addgene  ...to many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the... a resident endoplasmic reticulum (ER) protein instead localizes to the cytoplasm? Or more troubling, ...approximately 40% of human (and most eukaryotic) proteins localize to chemically distinct subcellular environments...

...incorporated into newly synthesized proteins instead of methionine. The labeled proteins can be detected using click-chemistry...turnover of individual proteins in situ.  For the visualization of specific proteins in situ immunocytochemistry...audiences. Right now, she is a science instructor at The Innovation Institute in Boston. When Eugenia is not ...protein is decreased in Alzheimer’s disease patient’s brains. However, it is not known why or how this happens...degradation. These processes control the abundance of proteins in cells. Homeostatic regulation of protein abundance...type. However, we cannot distinguish new from old proteins within that pool. Therefore, it is not possible...used to facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed...

...were at institutions focused only on tracking hours, and 22% of postdocs were at institutions where no... of some public institutions) to cancel plans to raise salaries. What are institutions doing after the...planning unionization at their institutions. One clear message that institutions have sent, with the lack of...their institution is doing from Future of Research over social media, not from their institution), is ...raise were: postdocs at institutions like Stanford and the Whitehead Institute, where salaries had already...street from these institutions) are raising salaries. Postdocs at the Salk Institute in La Jolla California...This post discusses how a nationwide injunction against the FLSA is affecting universities' decisions to...

...interest. Tagged proteins are typically expressed at much higher levels than endogenous proteins. Data obtained...Immunoprecipitation (IP) uses immobilized antibodies, or immunoglobulins, to isolate a specific protein out of a complex...study how a target protein interacts with other proteins or nucleic acids. Read on to learn more about ...optimized strategies for differing amounts of target proteins or antibody bonding strengths.    . Steps...from cells or tissues contain a complex mix of proteins seen as green squares, purple circles and blue...collected by centrifugation or a magnet. 4) Unbound proteins are washed away. 5) The target protein is eluted...eluted.     In the first method, an antibody against a target protein is immobilized, or tethered, on...