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  2. Which Fluorescence Microscopy Technique is Best for Me?

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    Blog Post
    ...microscope to use for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? ...? 2) Is the sample thin (< 15 μm) or thick? These questions will place each sample into one of four categories...capture in these types of samples is their fast movements. Depending on the sample, a temporal resolution... fluorophores higher in the sample. TIRF is also very gentle on the sample as the majority of the laser...static samples Ex: Fixed tissue sections (> 15 um), 3D cultures, and cleared tissues These samples always...onto a camera for visualization. Illuminating a sample in the opposite direction to fluorescence collection...plane within a relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner...

  3. A Control for All Seasons

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    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...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... tau hyperphosphorylation. You could run brain samples from both groups on a western blot and look for...

  4. What’s in Your Tube?: A Quick Guide to Measuring DNA by Spectrophotometry

    Type
    Blog Post
    ... easy to measure sample concentration and purity using only a tiny bit of the sample itself — so easy ...instrument can calculate a sample’s nucleic acid concentration based on the sample’s absorption at 260 nm (...spectra of a mixed sample.  But, other molecules that may be present in the sample also absorb light...levels exceed 70% of the sample content (Loughrey and Matlock, 2016). So, even samples with “good” A260/280...nm, you may have phenol in your sample.  RNA contaminants DNA samples can also be contaminated with RNA...especially problematic in low-concentration samples (samples with concentrations < 10 ng/μL), where absorbance... how much plasmid DNA you have or how pure your sample really is?  To assess the concentration and purity...

  5. Great Results Start with Great Standard Curves

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    Blog Post
    ...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...standard, standard dilutions, and unknown sample(s) in the same buffer if possible, or very similar buffers...their bicinchoninic acid assay (BCA) to normalize samples before running a western blot. Standard curves ... to extrapolate the concentration of an unknown sample. For example, a BCA assay uses a serial dilution...control protein, incubated alongside an unknown sample with the BCA reagent. The absorbance of both is...you to determine the concentration of the unknown sample.  How do I make the standards for a standard curve...standard curve by serially diluting a known control sample, called a standard. The ideal standard curve has...

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

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    Blog Post
    ... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...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...capture antibody that binds to the antigen in a sample and then a second detection antibody binds. The...

  7. Antibodies 101: Multiplex Immunofluorescence

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    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...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 ...referred to as immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC)...where an antibody binds a target protein in your sample, then a fluorescently labeled secondary antibody...

  8. The Power Behind NGS Plasmid Validation: seqWell

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    Blog Post
    ...carrying sample-specific barcodes. Rather than continuing to process 100s to 1000s of samples individually...projects in many labs. However, the high costs of sample preparation for high-throughput next generation...technology overview For any NGS experiment, the DNA sample being sequenced must be sheared into smaller fragments...workflows are limited by costly library prep, as each sample requires an individual library. The plexWell™ workflow...library prep”, in which a relatively large number of samples are subjected to a DNA-barcoding step that uniquely...labels DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create...procedure, the quantities of library molecules for each sample remain highly normalized across a wide range of...

  9. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...multiple proteins relate to one another in the same sample. For this strategy to work, it is important to...you use will depend on the target and sample type. Sample Preparation This step can involve multiple...target and sample type. Primary Antibody This is the first antibody applied to your sample, which will...of cells found in my tissue.” It is based on the same principles as other antibody-based assays, like ...fluorescent molecules are applied to a cell or tissue sample where they bind to their targets, most commonly...antibodies to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case...antibodies made in different species on a single sample, you can then use the corresponding secondary antibodies...

  10. Antibodies 101: Flow Cytometry Controls

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    Blog Post
    ...It will be from the same host species, have the same Ig subclass, and be in the same fluorophore (e.g.,...controls An isotype sample is prepared very similarly to a FMO sample: you make a sample from a combined ...your samples. The single color controls should be used as a starting point, to preserve your sample, and...experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well...controls will help you save on sample volume, so you can use most of your samples for flow analysis rather ...various controls to help you set up and analyze your samples. While you are probably familiar with the basics...controls (Figure 1A), instead of using your actual samples. They may also be used as a starting point for ...

  11. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    ...with each sample. Successfully analyzed samples will display the following parameters: Sample - The unique...multiplex sample, ICE includes visual representations of all detected edit types in the sample. The visualization...the ICE software has two analysis formats: sample by sample analysis, which can compare up to five editing...results and a list of the analyzed samples (see below).   If the sample run had no issues, the analysis ...perform more in-depth analyses on each sample by clicking on the sample name or on its corresponding bar graph...file. On the plot, the green (edited sample) and orange (control sample) lines should be close together before...files and provide basic information such as your sample names and guide sequences, and ICE will do the ...

  12. Antibodies 101: Flow Compensation

    Type
    Blog Post
    ... your experimental samples In order to do compensation on your experimental samples, you will first need...experimental samples. Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to...Compensation example based on experimental sample. Flow plots showing a sample before (A) and after (B) compensation...apply to your experimental samples as they did with your single control samples: if the population needs...fluorescent channel to another. When you label your samples with multiple antibodies, the fluorescent probes...general, if two fluorescent markers are read by the same laser, they will need to be compensated for one ...cytometer reads out the fluorophores FITC and PE on the same laser, you will need to compensate between the two...

  13. Behind-the-scenes of the Isolation of the Thermostable IgnaviCas9 From a Yellowstone Hot Spring

    Type
    Blog Post
    ...highly complex hot spring sample. The sample was separated into smaller samples of 5-10 cells each for sequencing... Mork collecting samples at the hot spring. Hiking all day and processing samples by night was fun...leave your samples behind! Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S ...biological dark matter and so the lab organized a sampling trip to Yellowstone National Park (YNP). Extremophiles...from these extreme environments. Gearing up for sample collection at the hot springs My husband, Paul ...post doc in the Quake Lab, volunteered to do the sampling and I happily agreed to accompany him to YNP the... and two deep cycle batteries for power so the samples could be kept frozen after collection. Figure...

  14. Troubleshooting and Optimizing a Western Blot

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    Blog Post
    ...time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...protein-based buffer (FBS, milk, etc.) against the same amount of sample.  If you’re using chemical blockers, you...gradient, compare your diluted samples. If multiple dilutions are showing the same signal (Figure 3B), that ...of protein in your samples. If you have a wide variety of protein amounts on the same blot, you may want...gradients. Protein gradients use dilutions of your sample or protein of interest to understand the dynamic...reagent gradient. In this approach, you’ll run the same protein concentration in multiple lanes and cut ...first step in any western blot is to lyse your samples. There are a few issues that can arise in this ...

  15. Antibodies 101: ChIP

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    Blog Post
    ...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...Homogenization for a tissue sample can also need significant optimization for the sample type and size.) It can...process is very similar for both, though tissue samples do require an extra homogenization step. For this...to focus on the ChIP process and not the upsteam sample collection process or the downstream analysis process...itself begins at...  Crosslinking Once you have your sample, the first step is to ensure your protein:DNA complexes...likely to remain attached to the DNA throughout the sample processing and therefore will require crosslinking...Typically, this can be done by cutting the tissue sample up and then processing with a homogenizer, but ...

  16. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...we just made the sample bigger? In ExM, and its various derivatives, biological samples are enmeshed in...label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link...the number of specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of... its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion...gel components to polymerize. Step 3: digest the sample to allow for even expansion.  In some cases, labels... ExM process allows you to physically magnify a sample so you don’t have to optically magnify quite so... no exception. First, expansion requires fixed samples. This limitation may be the both most obvious and...

  17. Antibodies 101: Immunoprecipitation

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    Blog Post
    ... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases..., incubate the sample as you would in an IP using a nonspecific antibody from the same host species as... from the sample prior to beginning the IP.  During the IP, the target protein in the sample binds to ...parallel with both a sample known to express the protein of interest, such as a sample transiently transfected...out in one of two ways. Both methods utilize the same basic strategy: immobilize the antibody or antibody-target... if the antibody used for IP is derived from the same species as the antibody that will be used for detection...protein/antibody interactions To prepare for an IP, samples are typically lysed in a non-denaturing buffer ...

  18. Deep Dive: Statistical Tests (Comparisons)

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    Blog Post
    ...comparisons. Paired and unpaired t-tests Samples that come from the same subject or population - before and ...referred to as two-sample or independent, but these designations all refer to the same test. On some occasions...five-sample data set yields statistical significance, you won’t know if only two of those samples are ...appropriate to use if all sample comparison is needed, without a high number of samples (~5). Tukey’s test ...you are solely interested in whether or not two samples are statistically different than one another, a... direction (i.e., you just want to measure if a sample is greater than a control), then a one-tailed test...assesses the sum of differences between pairs). Samples arising from different subjects or populations ...

  19. Lab Automation at Addgene

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    Blog Post
    ... from many bacterial samples at the same time as opposed to preparing each sample individually. Enter ...easy to spread out your samples. This can be useful if you’re transferring samples from 96 wells plates ...developed a protocol to run up to 192 samples at a time. It follows all the same steps and uses the similar buffers...device fits on the deck, and it’s easy to pipette samples in and out of it, you can set up a procedure for...compared to doing about 50 transformations in the same amount of time by hand! Setting up a basic protocol...buttons you can quickly tell the Biomek FxP how much sample to pick up, where to move it, and how long to keep...mobile pods - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod...

  20. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

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    Blog Post
    ...only antibody option is from the same species as your tissue samples. The bad news: this situation is ...excess secondary with serum from the same species as your primary/sample, and then applying this mixture to...from the same species and 2) these fragments will be more likely to bind (and block) the same epitopes...processing and sample preparation can also help reduce the levels of endogenous IgGs in your samples, but know...The culprits are endogenous IgGs in your tissue sample. Most common IHC protocols use two antibodies -... the first antibody. When endogenous IgGs of the same species as your primary antibody are present, your...staining (Figure 1A). Not all tissues will have the same levels of endogenous IgGs, so some researchers can...
Showing: 61 - 80 of 611 results