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We narrowed to 356 results for: SAM activators

Showing: 301 - 320 of 356 results

  1. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C....performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal...solution will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate...plate for individual cells, which should be on the same focal plane. Scan the entire plate and make note...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines...

  2. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...spatiotemporal control of transgene expression or activity can be achieved using switches, including riboswitches...function like your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb)...systems, which use two or more AAVs to transduce the same cell, can accommodate larger, modular genetic payloads...efficient AAV-PHP.eB, developed and deposited by the same lab. AAV-PHP.eB is the most commonly requested systemic...off-target organs. AAV.CAP-B22 was developed through the same process as AAV.CAP-B10 and shows high transduction...

  3. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...with samples, eliminating them from the procedure removes one potential source of cross-reactivity.   While...use the same secondary antibody for any ELISA protocol that uses primary antibodies of the same isotype...-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell...used to quantify the level of target antigen in a sample. While Engvall et al. originally developed the ... proteins and small molecules from a variety of sample types (Engvall 1971). Scientists value ELISAs for...using the method. ELISA basics All ELISAs follow the same overall process. First users coat a multiwell plate...

  4. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    ...presence of several biomarkers together in the same tissue sample predicts a certain cancer and want to look...you will need to start with a fixed sample. Depending on the sample type, this might be frozen or formalin-fixed...you image the first target, you inactivate the fluorophores in the sample, either by chemical treatment ...al, 2022 for a sample protocol.)  Simultaneous IF Depending on your needs, your sample, and targets, you...known to be abundant in your samples (with a primary antibody that has the same species and isotype as the...set of samples where you label only one target in each. Compare these individually-labeled samples with ...the sample is being damaged by all the handling steps, or there’s some unexpected cross-reactivity. Keep...

  5. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    ...than generally reactive ones. And make sure to check for any reported cross-reactivity when planning your...minimize the risk of cross-reactivity. To further limit species cross-reactivity, try to find secondary antibodies...cross-reactivity. For example, if you are using mouse IgG2a and rat IgG1 antibodies in the same application...specific secondary antibody, rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse...Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies. Only a fraction...of the antibody molecules present in a broadly reactive secondary antibody will bind to a monoclonal primary...different isotypes and subclasses, use a broadly reactive secondary antibody to increase the chance of primary...

  6. CRISPR Guide

    Type
    Guide
    ...helper activators; highest levels of single-gene activation (Figure 9D) In bacteria, activating gene expression...nuclease activity Cas9 enzymes will likely improve as more key residues for specificity and activity are discovered...-NG - NG; increase in vitro activity SpG - NGN; increase nuclease activity SpRY - NRN/NYN, where R = A...select for gRNAs with high on-target activity and low off-target activity. Knockout libraries often target...become a flexible tool to activate, repress, visualize, and isolate genes. Activation or Repression of Target...transcriptional activator (e.g. VP64) or repressor (e.g. KRAB; Figure 9A). Other examples of activation systems...instead of a two-component system (Figure 9C) SAM activators - co-expression of dCas9-VP64 with a modified...

  7. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...two different shRNAs that target the same gene can produce the same phenotype will alleviate concerns about...plasmid in mammalian cells. sin 3’LTR 3’ Self-inactivating long terminal repeat. f1 ori f1 bacterial origin...boiling water. If using a PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room...Biotechnol . 1997. 15(9):871-5. PubMed . Self-inactivating lentivirus vector for safe and efficient in ...

  8. Optogenetics Guide

    Type
    Guide
    ...different colors of light to activate/silence the same neuronal population, or activate different neural populations...respectively. Color of activation light. There are a variety of different activation wavelengths, ranging...including protein activation, membrane localization, and transcriptional activation. In the widely used... transcriptional activation domain and DNA binding domain, allowing light to activate transcription. Phytochromes...Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike ...the closing of the channel 470(activ.), 590(inact.) ReaChR Red-activatable variant of CrChR2 590 Channelrhodopsins...Different activation wavelengths also make it possible to combine multiple opsins in the same experiment...

  9. A Control for All Seasons

    Type
    Blog Post
    ... replicates are repeated measurements of the same sample and are included to assess variability in the... the tau western blot several times with the same samples and compare the results across runs. If the ... wild-type and knockout samples. In this test, wild-type and knockout samples are treated with the identical...present in the wild-type samples and absent in the knockout.  If knockout samples are unavailable, try substituting...control, and cross-reactivity control when multiplexing. A, wild-type and knockout samples are the gold standard...interactions between the sample and the secondary antibody. C, a label control treats the sample to the full protocol...interpretation of results. A positive control is generally a sample or group that will have a desired response. For...

  10. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    ...recognize the same region, or epitope, of the target protein. These antibodies are also all the same isotype...heterogeneous mixture of many antibodies that recognize the same protein. In the immune system, antibodies are produced...peptide sequence derived from this protein. At the same time, an adjuvant such as KLH or Freund’s adjuvant... without causing high background due to cross-reactivity. When to use polyclonal antibodies One advantage...clones will still recognize your protein. For the same reason, they’re also useful in experiments where...experiment, you should either order several vials of the same lot of polyclonal antibody, or select a monoclonal...your polyclonal antibody, you’ll want to use the same positive control across experiments to be aware ...

  11. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...because the pair belong to the same isotype and will react with the same secondary. If you were to change...the indirect method since a secondary antibody reactive to a certain isotype will bind to all members ...that group. If you use two antibodies that are the same isotype then the secondary antibody will bind to... Figure 2: Two primary antibodies with the same isotype can not be used for multiplexed indirect...parental species which can be bound by broadly reactive secondary antibodies to that parental species....

  12. Lentiviral Vector Guide

    Type
    Guide
    ... and actively dividing cell types, whereas gamma-retroviruses can only infect mitotically active cell ...packaging plasmids. tat in trans Trans-activator; binds TAR to activate transcription from the LTR promoter...the transfer plasmid, and this requires Tat for activation. Second-generation plasmids include (Figure 3...lentiviruses most often integrate into transcriptionally active regions, regions recently involved in translocation...Instead of selection by an antibiotic, fluorescence-activated cell sorting (FACS) is used to sort cells expressing...CRISPR libraries, including knockout, inhibition/activation, and prime and base editing. Browse Addgene's... systems do not require the HIV protein Tat to activate the promoter on the transfer plasmid. Many of ...

  13. Protocol - How to Design Primers

    Type
    Protocol
    ...must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding a nucleotide...primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30...

  14. Gamma-Retroviral Vector Guide

    Type
    Guide
    ... and actively dividing cell types, whereas gamma-retroviruses can only infect mitotically active cell ...are found in proto-oncogenes. This can lead to activation of the oncogene and possible development of cancer...Instead of selection by an antibiotic, fluorescence-activated cell sorting (FACS) is used to sort cells expressing...site selection is driven by strong enhancers and active promoters. Nucleic Acids Research , 42 (7), 4257... (unique 3’) contains sequences necessary for activation of viral genomic RNA transcription. R is the ...cis Unique 3’; contains sequences necessary for activation of viral genomic RNA transcription. Removal of...Both lentiviruses and gamma-retroviruses use the same packaging genes. However, they are different viruses...

  15. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...compare different clones of the same target to see if the results are the same/similar, emphasizing the antibody...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...confirming it will produce the same results even to a different sample. Finally, as a lab we discuss the...underlying causes of this unreliability: cross-reactivity (binds to non-target proteins), variability (...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...

  16. Antibody Guide

    Type
    Guide
    ...fluorophores are available in a range of colors activated by different wavelengths, allowing for easy differentiation...conjugated with HRP. Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane...antibody, followed by incubation and wash steps. Activate the conjugate and read the output via a plate ... antigen and the conjugate, typically HRP, is activated and its output measured. This method can be performed...secondary antibody is added. The conjugate is activated and the output is read. A standard curve with ...deposited into tubes or plate wells) by fluorescence activated cell sorting (FACS). Figure 7: Flow cytometry ...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...

  17. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...particles = number of particles per mL of sample. The purity of the sample can be estimated by looking at the...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...Trapnell. "Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy." Journal...

  18. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...known titer on every qPCR run. Note that the rAAV sample has to be digested with DNase before undergoing...Grimm, D., Kern, A., Pawlita, M., Ferrari, F., Samulski, R., & Kleinschmidt, J. (1999). Titration of AAV...Harvey, B. K. (2017). Assaying the Stability and Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy..., J., Hennigs, J. K., & Körbelin, J. (2021). Inactivation of Adeno-Associated Viral Vectors by Oxidant-Based...

  19. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...thiols, to cleave disulfide bonds. Load the samples, one sample per lane, onto the top of a resolving gel...all of the proteins in a sample migrate through the stacking gel at the same pace and enter the resolving...consistent size and density present in all samples. Uneven sample loading or incomplete protein transfer ...Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated in a gel, transferred...is the first step of a western. To prepare the samples for SDS-PAGE, measure the protein content and normalize...normalize to ensure equivalent loading. Denature the samples to their primary amino acid sequence by boiling...crosslinked polymer acrylamide. In addition to the samples, a protein standard of known molecular weight in...

  20. Antibodies 101: Validation

    Type
    Blog Post
    ...antibody signal in the retinal sample, but little to no signal in the liver sample, which you do (C), so your...epitopes on the same target reduces the likelihood that both antibodies would bind to the same off-target ...core goals of antibody validation:   Confirm reactivity with the target antigen - Recognition of an immunogen...to - particularly when you start using complex samples such as whole cell lysate or tissue sections. It...require variable expression of your target across samples - it is hard to distinguish between specific, invariable...there is a wealth of transcriptomic data from this sample type, so you use your bioinformatics skills (or...Removing (or reducing) the protein of interest in your sample, should lead to reduced or eliminated antibody ...
Showing: 301 - 320 of 356 results