...Direct staining is quicker than indirect staining and eliminates potential non-specific staining that may...its native state.  Antibodies-based flow cytometry can use both direct and indirect staining methods. In...intracellular staining where binding of large immune complexes may be hindered. Indirect staining can be beneficial...fixation. If staining both cell surface and intracellular proteins in parallel, then stain the surface ... non-specific staining.  If you’re using antibodies for detection, include an unstained control with cells...cells that went through the staining procedure minus the antibody. An unstained control allows you to detect...-specific staining associated with the primary antibody. When following an indirect staining protocol,...

... electrons are elevated from a ground state to an excited state through the absorption of photons. After... elevation in this excited state, the electron returns to its ground state by emitting a photon with slightly...lengths of time, the stability of fluorescence over time is crucial. For instance, the fluorescence of...toxicity and high photostability is crucial. These properties ensure a more consistent and stable signal over...Fluorescence refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic...al., 2014). Other dyes may prove challenging to stain intracellular compartments in in vivo model systems...so extra care must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility...

...which heavy chain constant domain it pairs with its variable chain domain. B cells start by co-expressing...expressed with the IgM and IgD constant domains because they are the first constant domain genes in the cluster...heavy-chain constant genes μ and ẟ. These are the first two genes in the heavy-chain constant domain gene...heavy-chain constant domain genes, placing the variable domain genes next to a different constant domain gene... antibody that’s determined by its heavy-chain constant region (see Antibodies 101: Introduction to Antibodies...antibody isotypes that each have a unique heavy-chain constant region: IgM, IgD, IgG, IgE, and IgA.    ...labeled with Fc, Fab, heavy chain, light chain, constant region, and variable region.   Why are antibody...

...reporter. The only caveat is that HA staining is weaker than reporter staining. Picking the right reporter for... them. HRP RANbody staining results in a ≥10-fold increase in signal over staining with a primary/ labeled...antigen with a standard primary/labeled secondary Less sensitive than HRP-RANbody staining Requires secondary... IgY antibody Staining cultured cells and tissue sections Great for multicolor staining of tissue since...antigen with standard primary/labeled secondary   Less sensitive than HRP-RANbody staining Requires secondary...secondary antibody staining GFP, RFP, H2A2B   Are you ready to start making your own RANbodies? Great...tissues. Although antibody production is well established, it’s also a process that’s difficult for individual...

...photoswitched from a dark state to a bright fluorescent state (“kindling”), and from a bright state to a dark non-...be activated from low fluorescent states to high fluorescence states, they can change from one fluorescent...switched on from a state of dim or no fluorescence, to a brighter fluorescent state by exposure to a specific...Padron is photoconverted from a dark state, to an activated state, emitting bright green fluorescence.... and ability to switch from a dark state to a red fluorescent state make them useful for 2-color imaging...parameters to consider, such as brightness, photostability, pH stability, chromophore maturation rate at 37 °... monomeric) and display bright and photostable green and red states. Beyond these imaging applications...

...cancer development, metastasis, and drug resistance, Bhang says “the [drug] resistance story panned out ...Using ClonTracer to study drug resistance Although Bhang and co-workers started using the tool to look at ...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...majority of the resistant populations. In fact, 88% of the barcodes within the resistant populations were... populations with resistance to both drugs. The future of ClonTracer Since starting her new role as an...determine whether or not cancer cell populations resistant to drug treatments arise during treatment or are...preexisting in the parent population prior to the start of treatment. They hypothesized that, if they labeled...

...Amplification Efficacy Assumptions Standard Curve Yes Standard Curve No experimental sample variation...variation ΔΔCT No Standard Curve reference=target Pfaffl et al No Standard Curve sample=control...deep dive series, which aims to increase your understanding and technical proficiency with common applications...in protein expression. (If you're just getting started, check out our Introduction to PCR.) But if you're...you're ready to go beyond the 101 and take your understanding to the next level, read on for more in-depth... differences of twofold or greater with good statistical significance (Taylor, 2017). With the right conditions...sometimes cause as many issues as it solves, so understanding and using the application correctly is imperative...

...improve resolution, use a stacking gel on top of the resolving gel. A stacking gel typically has a different...antibodies used for staining. To address this, the membranes are incubated before staining in a buffer containing...separated in a gel, transferred to a membrane, stained with antibodies, and finally visualized.   ...modifications, quantify protein levels relative to standards, detect the cellular location of proteins, and...acrylamide. In addition to the samples, a protein standard of known molecular weight in a separate lane can...separate by size. Smaller proteins encounter less resistance in the acrylamide matrix and migrate faster through...percentage of acrylamide to increase migration resistance and improve separation. For large proteins, reduce...

...the following paths to maintaining a status in the United States including green cards that are well-tailored...relied upon for legal advice in any particular circumstance or fact situation.  The information presented...looking to continue their careers in the United States. There has been a growing shift away from employers...with filing a permanent residence case with United States Citizenship and Immigration Services (USCIS). If...with working with US immigration in its current state. As of September 2017, USCIS received 116,224 employment...government sectors.  Within these sectors, you could start your own company, work in a bench research career...plan for and develop your career into a series of stages to refine and build skills overtime for success...

... gold standard to demonstrate primary antibody specificity. B, secondary antibody-only staining will allow...Such species-on-species staining can be partially addressed with stringent staining protocols and the use...omitted from the staining but all secondary antibodies are included. If, for instance, you omitted a mouse...and outs of controls for biological experiments, starting with general controls and then moving on to controls...applications. After reading, you will have a better understanding of the different types of controls and be able...groups were run on a western against a protein standard (P) to check for tau molecular weight shifts. ...the precision and accuracy and therefore the statistical power of an experiment. Technical replicates ...

...colleagues identified a highly bright and stable fluorescent protein, StayGold. Although it quickly saw wide interest...named QC2-6 FIQ or mStayGold (Ando et al., 2023). This variant has similar photostability and brightness ...FocalPlanes piece on mStayGold inspired and informed this section.  Find mStayGold plasmids at Addgene .... (2023). Bright and stable monomeric fluorescent proteins derived from StayGold. ResearchSquare (Preprint...Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer...37095348. Breaking up is hard to do: Monomeric StayGold variants By Mike Lacy In 2022, Atsushi Miyawaki...dimerization without losing that brightness and stability. Here’s a quick overview of these new variants...

...outside of your department can understand your cover letter and research statement. My personal litmus test ... sentences) Simply stated, what are your aims? You could directly state your official aims, but ... to Get a Faculty Position. This edition has substantial expansions on the chalk talks and Zoom interviews...sections, along with new thoughts on research statements. Snapp, who is always looking for ways to improve...helpful. All went on to get faculty positions at top state colleges, private universities, and medical schools...publications and grants. 2. Write succinct and easy to understand documents A common misconception is that search...get to the punch line is a terrible sales pitch. State in the first sentence or two what your research ...

...development of drug resistance in bacteria, the tumor cells that escape the elimination stage acquire genetic...favorably attaining the equilibrium stage. This can be called “stalemate equilibrium” where the cancer stops...cancers differ in practically every facet starting from manifestation to their specific interactions with different...the immune mechanisms of the host. The cause, manifestation, diagnosis, recurrence, and treatment of cancer...the acute immune responses that eliminate early stage cancer cells, chronic inflammatory responses can... tumor cells spreading to other organs during metastasis. Evading Recognition Tumor cells over-express...damage healthy cells, and often cannot eradicate metastasis or eliminate the probability of a relapse. Chemical...

...). Taq polymerase is thus a heat resistant enzyme that can withstand changes in temperature. Taq was first...the starting template DNA the fluorescent signal throughout the reaction is compared to a standard curve...detected compared to the standard curve can be used to determine the amount of starting material in your sample...Chemistry for his work. Since then PCR has become a standard and essential practice in molecular biology and...heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic bacterium...range in which the forward and reverse primers are stable. At this temperature the primers can anneal to ...stranded DNA template strands. DNA polymerase is also stable at this temperature and can bind to the primers...

...comparison of this one-step staining with nanobodies vs two-step staining with antibodies. Nanobodies...before application to the sample. Staining with this multi-color staining workflow yields similar localization...localization patterns as cells stained for one target (compare nanobodies staining in figure 4A vs 4B). How...structure, HCabs are like a pared down version of a standard IgG. See below for a comparison of nanobody, HCab...double-edged sword. An antibody’s large size creates more distance between the target antigen and the fluorescent... fusion with a reporter enzyme. For multicolor staining, careful consideration must be used when selecting...different IgG subclasses of 1° for multi-color staining Can be expensive Nanobodies   Recombinant...

...usually started to spread (metastasize) and turned into Stage 3 or Stage 4 cancer. These later stages of a...indicate, for Stage 3, that the cancer is large in size and may have spread while Stage 4 indicates that...types of cancer have different challenges and obstacles to overcome, mesothelioma sufferers can see great...first exposure to asbestos to the time when someone starts seeing – or feeling – the first mesothelioma symptoms... time. Doctors and researchers don’t entirely understand why some people develop the disease while others...do not work very well. The best option in such instances is palliative care, which helps patients manage...discover the cancer earlier, in particular before it starts to spread beyond its point of origin. One of the...

...Fluorescent DNA staining is another DNA staining alternative. This method uses DNA stains like DAPI and ...the dry state for a longer time. When they come in contact with a nutrient source, they start proliferating...guest blogger, Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute of Biosciences and Technology.... The absence of a rigid cell wall makes them resistant to antibiotics and antibacterial drugs like penicillin... human scalp, sneezing, coughing, etc. Also, a constant in-flow of individuals wherever the cell cultures...this task, you can use PCR based detection, DNA staining, fluorescent tagging, agar plating, etc. Be wise...Culturing on agar plates/broth is considered the ‘gold standard’ for detecting mycoplasmas. In this method, the...

...for your standards (ladder). You’ll want to select a pre-stained or unstained set of standards that covers...But if you understand the technical aspects of a western blot, you'll be able to understand how to modify...have enough lanes available for your standard curve, your standard, and your sample(s), as you can only...methods and buffers to choose from. If you are starting from tissue, you’ll likely want to use a mechanical... V, 1–2 hours Good separation, quality, and stability Limited protein size range Tris-acetate ... visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and...you’re not running a western. They are not very stable and will noticeably degrade after several months...

...enhancer sequences in different chromatin states. Alexander Stark and colleagues at the Research Institute...their position relative to a transcription start site. In STARR-seq, randomly sheared genomic libraries ...(Q3) MARCH 2016Enhancer activity mapping with STARR-seq   Although many technologies have been developed...Pathology have developed an alternative method, called STARR-seq (self-transcribing active regulatory region ...over input levels using next gen sequencing. The STARR-seq backbone is available from Addgene in both human...including but not limited to cell growth and drug resistance. Recently, the Wu Lab at Texas Tech developed...plasmid or as individual plasmids shipped as agar stabs. Iverson, et al. ACS Synth Bio. 2015. PubMed PMID...

...produce substances toxic to themselves? What are toxin-antitoxin systems? These toxic substances are part...these systems is that the toxin is stable while the antitoxin is unstable, meaning that the cells must continually...the pair is more stable than the other. Here, the restriction endonuclease is more stable than the methyltransferase...degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain the plasmid... contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked...) contained within it by selecting for Zeocin resistance. Then, counter-selection with methanol medium... unmethylated sites in the chromosome. The more stable endonuclease can then cut the chromosome leading...