...for other antibody topics.     References and resources References:  Björck L, Kronvall G (1984) Purification...elute and measure the target. However, the two different approaches allow for optimized strategies for ...amounts of target proteins or antibody bonding strengths.    . Steps of an IP: 1) Protein extracts... appropriate for most situations.  A slightly different method is desirable if the antibody is expected...Protein G have an affinity for immunoglobulins, the strength of the interaction varies between antibody host...than those of agarose beads, despite the size difference. Magnetic beads are particularly advantageous...many factors, including salt concentration, ionic strength and pH, affect binding the lysis buffer may need...

...virus:  Lowering cytotoxicity. Different envelope proteins have different levels of cytotoxicity and the...Altering sensitivity to serum. Different pseudotyped viruses are differentially inactivated by nonspecific...Table 1 (Cronin et al., 2005)     References and Resources References: Crawford KHD, Eguia R, Dingens AS...protocol for generating lentivirus, but you use a different envelope glycoprotein instead of the wild-type...review articles to find tables and information on different viral membranes, tropism, and advantages and disadvantages....084 Li Q, Liu Q, Huang W, Li X, Wang Y (2017) Current status on the development of pseudoviruses for ...

...host species. Created with BioRender.com. How are shuttle vectors different from other plasmids? If ...protects against any of them. But different aminoglycosides work on different host species: kanamycin targets... unrelated host species, even from completely different kingdoms of life. That’s convenient for researchers... Shuttle vectors have to address several big differences in the ways vectors are used across species: ...resistance gene on your vector might require two different selection molecules! It all depends on how the...Auxotrophic selection Another option is to use a different method of selection entirely. Auxotrophic selection...host range,” these terms are typically used in different (though overlapping) ways. Broad host range plasmids...

... can separate the DNA strands. Created with BioRender.com. Once DnaA breaks the local hydrogen bonds...Prokaryotic chromosomal replication. Adapted from BioRender template. Since DnaA binding is necessary ...proteins also contribute to this regulation in different ways: methylating newly synthesized DNA, blocking...numbers can allow RepA to link iterons from two different plasmids, forming a pair of “handcuffs” that blocks...RepA proteins form complexes between iterons on different plasmids in a process called "handcuffing," which...folding, blocking replication. Created with BioRender.com. Plasmids under stringent control of replication...was updated by Emily P. Bentley in June 2024.  References and resources Further Reading Quiñones-Valles...

...doping to forensics, DNA sequencing to drug testing. You can find Nick on LinkedIn. References 1. Skloot...Expression Attenuates Proliferation and Invasion in Renal Cancer Cells, Marker-independent identification ...the cell lines deposited with them and used by different researchers are free of contamination and match...from this bank. I am often asked, “What is the difference between materials available from commercial repositories...the same cell line from a colleague?”. The main difference is the QC and validation carried out by the commercial... The same test is so good that it is used as a forensic technique (aka DNA fingerprinting) that can place... produce a new batch once you’ve passaged your current batch too many times or you suspect there is contamination...

...are communicating across disciplines or doing different tasks. But these challenges can be exaggerated...equal opportunity to contribute. Using video conferencing tools like Zoom or Google Hangouts is a great...great way to include researchers from different locations. Having virtual face-to-face meetings as opposed... collaboration includes scientists working in different time zones, it’s important that everyone can access...project at the International Cancer Immunotherapy Conference in Frankfurt, Germany When funding and circumstances...techniques are being performed the same way at different sites. I did a lab exchange as part of the collaboration...allowed me to investigate my protein of interest in a French clinical cohort, neither of which I would have ...

.... The plasmids within it contain a variety of different promoters followed by leader sequences (encoding... at the BIOFAB showed that expression of many different genes was predictable based solely on the promoters...BIOFAB Plasmid Set contain terminators of varying strengths centered within GFP/RFP bicistronic reporters ...or protease sites to remove the epitope tags. Different plasmids in the kit can be used to purify proteins...prokaryotic expression in recent years, with many different systems now available. However, it can be difficult...ORF clones to allow testing and comparison of different cell-free expression systems; the Cell-free expression...bind specific RNA bases. By swapping out these different modular repeat segments, you can create a PUF ...

...what we need to know in order to even assess the current state of research became more clear throughout ...that, even though postdoc associations across different universities are trying to compile more comprehensive...path,” but individual institutions can have many different names for the positions commonly thought of as...the Starting Salary for Postdocs to $50,000 (the current median postdoc starting salary as reported by Paula...Student Career Outcomes University websites do not currently publish career outcomes for PhDs or postdocs. ...achieved. Of note, it was highlighted during the conference that just increasing funding for academic research... At Addgene we try to do our part in boosting awareness of various career opportunities through our career...

...microscopy and beautiful imaging.   References and Resources References  He, Y., Zhang, T., Sun, H. et al...killing the untransformed seedlings or callus (undifferentiated plant cell culture), which is great for a ...desired plants! PCR or fluorescent markers are currently used in these situations to select against CRISPR...such as cacti and beets. Betanin belongs to a different chemical family than the red/purple plant pigments...it for selecting transformation events and am currently planning to use it to simplify my work with an...(Bottom panels) RUBY expression can vary from different transformation events likely due to insertional...lower expressing RUBY plant is a pale pink in transparent tissues, or in green tissues is slightly darker...

...Why the HEK not? References and Resources References Shaw G., et al., Preferential transformation of ...determined it is most likely adrenal in origin. (Anatomy refresher: the adrenal gland is next to the kidney...were isolated from is still a mystery, but it’s currently assumed that the cells came from an aborted fetus...genetically female. HEKs are generally cultured as an adherent monolayer, though derivatives have been adapted...have some neuronal qualities.) While HEKs probably aren’t a great model of kidney cell biology, there are...

...of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these...genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering...system for multidirectional control The CRISPR interference (CRISPRi) model consists of a catalytically ... multiplex control via scRNA design is readily apparent in metabolic engineering. The authors elegantly.... cerevisiae by using three RNA programs to differentially alter the levels of five genes that encode ...sequentially rerouted the metabolic flux to generate differently pigmented derivatives of violacein. Using dCas9...that will still recognize guide RNA but with a different dCas9 binding module, alternative sets of transcriptional...

...available jobs, fields, and trends “Never Leave an Empty Seat” at conferences and seminars, as this is ... path to professorship. But recently a downward trend in funding and space in academic research labs has...traditional academic setting and to shape and strengthen interests and skills. Although the word “extracurricular...potential EAs and which professional skill sets are strengthened. This short list highlights the ubiquity of ...particular field of study Assess your interest in a different field Outreach activities (e.g. Volunteer...Marketing Communication Business models Conference or club organizer Management Leadership...know where graduate students and postdocs are currently getting hired? Let us know what topic you'd like...

...seeing adoption of the technology in different labs for different applications.” Li’s team noted that ... genetic diseases, and several treatments are currently being tested in clinical trials. It is speeding...cells of patients with cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison to other genome...for selection and optimization. In addition, the current work in developing anti-CRISPR proteins aid the...possible through the use of nanoparticles. [6].    References 1. Zhang B. 2021. “CRISPR/Cas gene therapy.” ...at genome editing with CRISPR-Cas9 technology.” Current genetics. https://doi.org/10.1007/s00294-019-01040...its Application in Genetic Diseases: A Review.” Current pharmaceutical biotechnology. https://doi.org/10.2174...

...of the program beneath it. You can jump to the different time stamps in the description to watch any sections...precisely. You can find a great discussion of different types of replication in Nosek and Errington 2017...another’s reagents with the confidence that they aren’t simply doing an experiment wrong. However, even... a plate or running a gel have intricacies that aren’t always mentioned in static written protocols. It...scientific findings. This makes sense on many different levels - it’s hard to process large amounts of...to get a publication (for better or worse, the currency for success in academic research). Initial positive...review our experimental designs and we need to be transparent about the reasons we alter these designs if we...

.... Unfortunately, there really aren’t any effective off-target reference controls. Off-targets and your...they do arise. Happy CRISPR’ing! References and Resources   References Rabinowitz R, Offen D. Single-Base...effects should impact your experimental design differently. Yes, it is always better to reduce off-target...mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq would ...acknowledged as a potential limitation in a study. Strength in numbers If you assess your off-target frequency...to control for off targets as well as clonal differences. If you are performing CRISPR library screens... as a transfection or transduction control, a different gRNA would have distinct off-target sites, eliminating...

...performance under different imaging conditions (e.g. different emission filters) or different set-ups. Many...uptake by cells. To correct for this variation, a reference fluorescent protein that is expressed at an equal...be used. An assay developed in our lab uses a reference protein that is translated from the same open-...between the fluorescent protein of interest and the reference protein (Goedhart et al, 2011) allows for a precise... a set of fluorescent proteins (with the same reference protein) under identical conditions, a ranking...for co-expressing mTurquoise2 with mVenus as a reference protein is available from Addgene. Another way...

...Nano-lantern technology. Nano-lanterns contain a Renilla luciferase variant fused to an FP; when supplied...Solving the problems of low luminescence By itself, Renilla luciferase has a low quantum yield, and when transiently... first Nano-lantern, a fusion of a mutagenized Renilla luciferase (RLuc8) with Venus. This luminescent...resonance energy transfer (BRET). Photons emitted by a Renilla luciferase variant are used to excite the fused...technique’s versatility and adaptability to many different contexts. Improving Nano-lanterns In 2015, Takai...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially... for excitation, you instead have to rely on differences in emission wavelength. Filtering the emission...

...imaging for advanced monitoring of different factors, in different or the same cellular location, all ...covered with packaging plasmids! References and Resources References  Bhalla N., Jolly P., et al. Introduction...biosensors provide quantifiable readouts for levels of different chemicals, hormones, proteins, etc., that you ...and zinc. These ion sensors can be targeted to different cellular locations or tissues depending on the...acceptor of a FRET pair on actinin will produce different signals based on changes to the structure of actinin...organisms, but AAVs make it possible. In AAV systems, different serotypes can be used to affect or determine viral...

...skills that were quite different from mine. Learning to bridge those differences was a pivotal moment in...technical problems in the lab, management involves a different skill set: mentoring, setting strategic goals,... day-to-day details. Ignoring individual differencesManaging a team isn’t “one size fits all”. Each team...team member has unique motivations, strengths, and learning styles. For example, during my time as a lab...autonomously. Failing to recognize and adapt to these differences could have led to frustration, micromanagement...cohesive, productive environment. For example, in my current role managing a cross-functional team on a biomarker...team meeting to highlight their complementary strengths. By encouraging alignment on goals and clarifying...

...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites...activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., 2018)...found in other species can recognize a variety of different PAM sequences. Researchers have also used experimental...specificity can target more sites. Created with BioRender.com. Specificity Specificity is the assessment...relative to eSpCas9 and SpCas9-HF across a number of different sites. Using a combined random and directed mutagenesis...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants ...