...Firefly Insertion of 5' promoter/enhancer regions Bert Vogelstein 12178 pIS0 Firefly Insertion of target... Renilla Insertion of target sequence in the 3' UTR David Bartel 12177 pIS2 Renilla Insertion of target...improved normalization. John Atkins 167576 mpCHECK2 Firefly/Renilla Insertion of 3' UTR sequences downstream...construct that contains your regulatory element of interest. ID Plasmid Description Gene/Insert PI Return ...Plasmid Collection You may also like... Fluorescent Proteins Blog: Luciferase 101 Blog: Technologies Enabled...organisms, ranging from bacteria to aquatic animals to insects. Luciferase catalyzes the oxidation of a substrate...elements on gene expression. Empty backbones for inserting regulatory elements before or after the luciferase...

.... 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 ...

...Plasmids 101: E. coli strains for protein expression Plasmids 101: Common Lab E. coli Strains Why Bacteria? E...Photoswitchable fluorescent proteins Various PIs Fluorescent protein fusion: contains empty vectors with N and...Expression: contains mEos2 with an N-terminal His tag. Photoactivable fluorescent proteins like this one... 3OC6HSL (Quorum sensing molecule) Cynthia Collins Contains luciferase inducible by the quorum sensing...factor bind to a particular sequence? Do these two proteins interact? What happens when I overexpress this...From recombineering to CRISPR , this collection contains a variety of tools to help you harness bacterial...binding sites Philippe Cluzel RNA localization: Contains a 2x array of the binding sequence for RNA-binding...

... 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...

... E MGC4638 INS insulin ILPR, IRDN INS-IGF2 INS-IGF2 readthrough transcript - INSL3 insulin-like 3 (Leydig...MGC119819, RLF, RLNL INSL4 insulin-like 4 (placenta) EPIL, PLACENTIN INSL5 insulin-like 5 MGC126695, MGC126697...MGC126697, PRO182, UNQ156 INSL6 insulin-like 6 RIF1 INSR insulin receptor CD220, HHF5 JAG1 jagged 1 (Alagille...communication between cells of the immune system. Interleukins Interleukins are cytokines that are secreted by immune... omega 1 - IGF1 insulin-like growth factor 1 (somatomedin C) IGF1A, IGFI IGF1R insulin-like growth factor...MGC18216 IGF2 insulin-like growth factor 2 (somatomedin A) C11orf43, FLJ22066, FLJ44734, INSIGF, pp9974 IGF2R...bA12D24.1.1, bA12D24.1.2 RLN3 relaxin 3 H3, RXN3, ZINS4, insl7 RNASE2 ribonuclease, RNase A family, 2 (liver...

...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...

...interactions as well as be used for both soluble and insoluble proteins, which can be a challenge for classic antibody...can take hours to label proteins and APEX2, although it can rapidly label proteins, requires H2O2 which ...interacting proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused ...interaction of two proteins while also biotin labeling and purifying additional proteins from a complex of... and an RNA component that contains an RNA motif of interest. Any proteins that bind the RNA motif will...interacting cell surface proteins with a biotin tagged LPETG peptide. Biotin tagged proteins can then be purified...There are approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells...

... the presence of proteins from samples containing a mixture of proteins. The proteins are separated based... Antibodies, also known as immunoglobulins, are ~150 kDa, Y-shaped proteins that are both a natural part...region. Two immunoglobulin (Ig) heavy chains and two Ig light chains make up each antibody molecule. The...the Ig heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable...Flow cytometry is used to detect proteins on the surface of or inside whole cells. Antibodies - typically...antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens such...such as parasites or microbes, or to proteins expressed on the surface of cells that have been infected ...

...Margaret Robinson’s lab, and more recently used by Stephen Royle’s lab, acutely depleted proteins from their...induction heterodimerization of proteins that contain rapamycin-binding domains, such as FKBP and FRB. The ...inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al., Dev Cell...light-oxygen-voltage (LOV) sensing domains from algae into chimeric Opto-RTKs. The LOV domains bind flavin cofactors...phosphoserine (SP218) insertion was moderately enhanced while two phosphoserine insertions (SP218/SP222) was... to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other markers...demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the genome. Although...

...utilized CRISPR as a powerful defensive strategy against viral invaders, and this system is proving to be...genome in 1987, but their function as a safeguard against bacteriophages was not elucidated until 2007. Scientists...re-exposure. More specifically, specialized Cas proteins snip foreign DNA into small fragments approximately...stretches of DNA known as CRISPR arrays. Separate Cas proteins then express and process the CRISPR loci to generate...system. Within the bacterial genome, a CRISPR array contains many unique protospacer sequences that have homology... potential target sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly... varying PAM sequences and number/types of Cas proteins. Makarova et al. ’s classification defines 5 types...

...loops at tetraloop and stemloop - Contains BbsI sites for insertion of guide sequences Addgene plasmids...2 and EF1a-zeo resistance marker. Contains BsmBI sites for insertion of guide sequences Zhang Lab SAM ... microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial...nickase) + CRISPR RNA array + tracrRNA: This plasmid contains three expression cassettes. To target a given ...NGG PAM sequence. 3. SpCas9 alone: This plasmid contains a single cassette expressing human codon-optimized...2. SaCas9 only: This plasmid ( PX600, #61592 ) contains a single cassette expressing human codon-optimized...SpCas9 Cas9: The PX551 plasmid (AAV-SpCas9, #60957) contains a single cassette expressing human codon-optimized...

...mix of proteins with a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane...albumin or other proteins. The sticky areas on the membrane will bind to the proteins in the buffer, blocking...labeled. Instead, a conjugated species-specific secondary antibody is used to visualize the proteins. In this...Introduction to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to...absence of a protein, but can also determine if proteins are being up- or downregulated in a system, detect... to standards, detect the cellular location of proteins, and can be a readout for protein interaction ...applied to the gel causes the negatively charged proteins to migrate toward the positive charge at the bottom...