...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

...electrocompetent cells is minimal (picogram range). It can be extracted from small bacterial cultures (i.e. 2-3 ml...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...

...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...challenges in 2-photon in-vivo imaging of voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  ...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec;5(12):3610-6.(1985). doi: 10.1128/mcb.5.12.3610...

...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...

...Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates,...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express...

...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...Pharmacology and Optogenetics. Cell 162.3 (2015): 662-674. doi: 10.1016/j.cell.2015.06.058. Pubmed PMID: 26189679...

...eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample type(s) Cell lines Cell lines, primary...Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...

...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction... we identified in step 2. The result is a heterogenous population of stem cells, usually housed in a single...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...

...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...synthesized inside of what is called the “target cell” (or host cell) by the viral protein reverse transcriptase...

...reviewed in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...as it would for a full-length viral genome (Figure 2). While it is technically possible to encode all four...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...choose cell lines that are highly transfectable.  The amounts of each plasmid transfected into cells are ...