...visualize multiple targets at once. An advantage of multiplexing is that you can ask how multiple proteins... sample type. Sample Preparation This step can involve multiple smaller steps that all help to ensure...and colors makes it easy to perform multiplexed IF. By using multiple primary antibodies made in different... maintains sample morphology with minimal impact to the target epitope. There are multiple methods for...methods you use will depend on target and sample type. Example steps include: permeabilization, antigen...target and sample type. Primary Antibody This is the first antibody applied to your sample, which will... and Imaging Similar to Sample Preparation, this step can involve multiple smaller steps, but the aim...

...detect or analyze complex edits, such as those generated by multiplexed delivery of multiple sgRNAs. Since...For a multiplex sample, ICE includes visual representations of all detected edit types in the sample. The...with each sample. Successfully analyzed samples will display the following parameters: Sample - The unique...indel.   For a complete guide to ICE analysis, including detailed explanation and examples, read the paper...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...

...emulsion droplet technology that fractions the PCR reaction sample into approximately 20,000 droplets. Each... our blog post “Droplet Digital PCR for AAV Quantification.”   Multiplex PCR Multiplex PCR, as the name...utilize multiplex qPCR to quantify the concentrations of multiple starting DNA templates in one sample. It...target DNA levels Multiplex PCR Amplify multiple targets from one sample Disease or pathogen identification... molecular diagnostics.  Digital droplet PCR, ddPCR  Digital droplet PCR or ddPCR is a method that provides...analyzed by a droplet reader, which measures the fluorescence amplitude of each droplet. The fraction ...method in which multiple targets can be amplified in a single PCR experiment using multiple primers all in...

...detecting specific populations of cells in a complex tissue or sample (1). When combined with cell sorters, ...information about the cell’s complexity. A cell with a high degree of internal complexity such as extensive membranes... of cells. During FACS, the sample stream oscillates to generate droplets that are charged as they pass...plate. Each droplet contains a single cell that is assessed for the desired parameter. A droplet containing...rule out dead cells and debris. For example, a whole blood sample will contain a mix of cells including...differences in their size and complexity, cells from a whole blood sample will separate into distinct populations...ensure that samples are single cell suspensions. For cells that tend to aggregate, pass the sample through...

...can test three or more groups (two samples and a control, for example) simultaneously, with up to two independent...differences exist between. For example, if an ANOVA analysis on a five-sample data set yields statistical...appropriate to use if all sample comparison is needed, without a high number of samples (~5). Tukey’s test ...positives. Control vs. all samples Oftentimes there exists a control group and multiple experimental groups,...test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal levels... variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric...They are classic parametric tests and one of the simplest statistical tests available. Tailed t-tests If...

...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...extrapolate the concentration of an unknown sample. For example, a BCA assay uses a serial dilution of a ...concentration of the unknown sample based on its measured response. An example of this is shown in Figure...their bicinchoninic acid assay (BCA) to normalize samples before running a western blot. Standard curves ...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...

... the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety...coated with a competing antigen (green).  The sample (purple) is preincubated with a reporter enzyme labeled...-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell...control, include a sample that is known to contain the target antigen. Some examples could be an endogenous...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...capture antibody that binds to the antigen in a sample and then a second detection antibody binds. The...

...protein:DNA complexes with antibodies specific to your protein(s) of interest. Once the complex is captured... Once you have your sample, the first step is to ensure your protein:DNA complexes will remain bound to...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...the protein:DNA complexes. Once you’ve finished fragmenting the DNA, spin down the samples to remove cell...protein:DNA complexes and finally (5) isolated the DNA from your antibody:protein:DNA complexes. Your DNA...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...

...structure of the complementary heteroduplex. Base mismatches cause bulges in the duplex that may not fit...helix, purple; sgRNA, dark blue.   The REC lobe binds tightly to the repeat:anti-repeat duplex of the...This cap is where the DNA:RNA heteroduplex transitions back to DNA duplex, and it’s the reason guide sequences... A RuvC loop caps the heteroduplex and marks the transition back to a DNA duplex. sgRNA, dark blue; target...structurally resemble any other known proteins; they are completely unique to the CRISPR system (Nishimasu et al....CTD, yellow; REC lobe, light blue; bridge helix, purple.   Apo Cas9 changes conformation frequently... these regions, even if they are present in the sample. Other techniques, like electron microscopy and...

...If your plasmid can be classified in multiple categories, please check all of the appropriate categories... a collection that is greater than 75 plasmids, please let us know by contacting [email protected]. ...information directly into the file. Email the completed spreadsheet to [email protected] along with ...plasmid can be added on subsequent pages. Type – please choose from one of the following descriptions for...used to knockdown expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid...through cloning. *Note — If you will be depositing multiple plasmids and would prefer to send us your plasmid...e-mail us at  [email protected] Special cases Please contact us at [email protected] or +1 (617)-ADDGENE...

...across the board. If the samples or sample conditions change, the multiplexed protocol may have to be ... If there are multiple amplicons, or primer dimers forming, the curve will show multiple peaks.    ...optimizing for LightCycler machines.) Multiplexing It is possible to multiplex qPCR when using fluorescent probe-based...individually run sample when developing the protocol and optimized until the multiplexed and individual ... With the right conditions and parameters, some people find it can detect even smaller differences, and... can be the more optimal choices at times. For example, chemistries that rely on hairpin design of probes...the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison...

... worked with people outside the department to accomplish a variety of goals. For example, I worked with...experts that complement your (scientific) skill set and is therefore very useful for complex projects. It...met interesting people at conferences and you have successfully reached out to people outside your organization...Meeting people outside your organization just to have a quick chat can be rather tricky (think people based...Addgene I work with people from various organizations in science and beyond. For example, I help organize...collaborations are becoming increasingly important as more complex approaches to scientific questions become popular...mutual benefit.  Our partnership with SnapGene, for example, allows us to display high-quality maps and annotations...

... can be completely denaturated (left) or complexes may dissociate  (right). B) Protein samples protected...protect fragile samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM samples can be damaged...and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered and evolved...antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure determination By Mike ...for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki Lab...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it easy...cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic...

...cells or tissues contain a complex mix of proteins seen as green squares, purple circles and blue triangles... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases...capture antibody. This will deplete the nonspecific proteins from the sample prior to beginning the IP....proteins are completely removed. Wash steps should be performed even with a pre-cleared sample.  Eluting ...parallel with both a sample known to express the protein of interest, such as a sample transiently transfected...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...

...experimental samples. Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to...Figure 2: Compensation example based on experimental sample. Flow plots showing a sample before (A) and after...channel to another. When you label your samples with multiple antibodies, the fluorescent probes on the...Compensation example with single color control. Flow plots showing an APC single-color control sample before... your experimental samples In order to do compensation on your experimental samples, you will first need...apply to your experimental samples as they did with your single control samples: if the population needs...overlap in spectra is often called "spillover." For example, if your flow cytometer reads out the fluorophores...

...your samples. There are a few issues that can arise in this step: protein degradation, incomplete lysing...time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...wells. Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill ...proteins going completely through the membrane or larger proteins not transferring completely onto the membrane...your protein gradient, compare your diluted samples. If multiple dilutions are showing the same signal (Figure...amount of protein/sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring...gradients. Protein gradients use dilutions of your sample or protein of interest to understand the dynamic...

...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...times, and the physical properties of the sample itself. For example, cultured cells are typically easier ...structure. For example, cytoskeletal structures that form continuous lines in pre-expanded samples can sometimes... popularity with an abundance of protocols and examples of applications from across disciplines now available...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...

... permeabilize a sample. But you can also use organic solvents after fixing your sample with a cross-linker...during each step. Now that you know the basic principles of IF, we’ll take a slightly deeper dive into...protocols. Here we’ll start with just the first couple of steps - mainly, fixing and permeabilizing. ...the point?  Fixation allows you to freeze your samples in a given state or point in time. You want to ...processes while maintaining the structure of your samples, the relationships between cellular components,... molecules and it is great for preserving your samples and maintaining cellular and tissue morphology....cross-linker for IF. However, you might come across a couple other terms that can cause some confusion. Paraformaldehyde...

...connections This is a site for meeting people. Use the settings to let people see who you are when you look at...has spam profiles and trolls. People don’t like to connect with complete strangers as it borders on my... a LinkedIn profile is absolutely necessary. If people can’t find you on LinkedIn, you will lose opportunities.... When I am speaking at a new institution, for example, I will often see a whole bunch of notifications... (unless they’ve chosen to hide it). While most people use LinkedIn messaging, not everyone likes to. ...opportunity to start things out right. Also, many people on LinkedIn don’t appreciate random requests without...results in a generic invitation. 3) Connect with people you have really talked to The best time is within...

...I’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot...each individual sample, this will reduce convenience and comparability because each sample with be quantified... low-concentration samples can also behave differently in PCR, even if both samples fall within the linear...reference sample of known titer, which is at a higher titer than the AAV RSM. By using these samples in all... titer values of new AAV samples. We also typically quantify our AAV sample by two qPCR assays and compare... viral vector preparation protocol, and we are pleased to report that over 95% of viral capsids in representative...Systems, Harvard University.    Which titer do people usually report? Addgene and other viral vector ...