...You’ve gotten the plasmid encoding your protein of interest from Addgene, transfected it into your target... keen to study is expressing in your cells? Immunoblotting or simply the western blot, or western, is ...Burnett et al., 1981).  Introduction to western blotting  In a western, proteins are: (1) separated by ...proteins to migrate toward the positive charge at the bottom of the gel and separate by size. Smaller proteins...increasing range of acrylamide content from top to bottom allowing a mix of proteins with a broad size range...gel and membrane are “sandwiched” together with blotting paper and an electric current is applied causing...this method.  Finally, commercially available dry blotting systems efficiently transfer a range of protein...

...the interview with Darrell Kotton Chapter 1: Introduction to Darrell Kotton and the Boston University ...place?" Many thanks to our guest, Darrell Kotton. Dr. Kotton is the David C. Seldin Professor in the Department...Darrell Kotton is the director of the Center for Regenerative Medicine at Boston University Medical School... Chapter 5: Research currently under way in the Kotton Lab. One of Darrell's favorite projects - differentiating... as an NIH-funded Principle Investigator in the Kotton Laboratory and is the founding Director of the ...Plasmids from the Kotton Lab Learn more about Stem Cells ... darrell kotton...

... https://doi.org/10.7554/elife.05826 Blum, M., & Ott, T. (2018). Xenopus: An Undervalued Model Organism...

...evolution. Southern blotting and quantitative real-time PCR Southern blotting, named for its inventor...southern blotting can be used to identify the presence of that mutated gene. In southern blotting, DNA is... look at three historical techniques: southern blotting, restriction mapping, and sequencing gels, as ...signal in comparison to a control sample. Southern blotting applications range from confirming the results... clinical diagnostics. One example of southern blotting being used to identify a gene mutation is the ...test used for sickle cell anemia (2). Southern blotting, in conjunction with restriction digest analysis...When a restriction digest with MstII and southern blotting are conducted on a sample from an individual without...

...detect proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ELISA is that...or by binding to an antibody that is coating the bottom of the well), and then its concentration is detected...technique works by first coating the wells of a flat bottomed plate with a capture antibody that is specific...amount of an antibody that recognizes it, while the bottom of the wells are coated with another protein — ...light absorbed by a solution - of these wells is plotted against the known protein concentration. Importantly... Figure 5: A standard curve is generated by plotting OD against known concentrations of your target...protein. The OD of experimental samples can then be plotted along this line to calculate the protein concentration...

...control can be accurately quantified In western blotting, the relationship between protein abundance and...detection range of the assay is between the two dotted lines. Created with BioRender.com. To see why...“the most common error associated with Western blotting quantification” (Gilda & Gomes, 2013). To address...covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI and loading control...measurement will take place either before or after blotting for your POI. Figure 6: Total protein measurements...single-protein controls in semi-quantitative immunoblotting. Journal of Neuroscience Methods, 172(2), 250...Addgene Blog Antibodies 101: The Basics of Western Blotting Technical Design of a Western Blot Troubleshooting...

...transduced with lentiCas9-blast. Anti-Cas9 Western blotting was performed on whole-cell extracts from stable...multiplicity of infection (MOI). Anti-vinculin Western blotting is a loading control. Pro-Tips: 1. It’s all...under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts from the ... Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9...good image. You could probably also do Western blotting against GFP expression, but I didn’t. 2. In the... I could even also see the 85 bp product at the bottom of the gel in the BRAF gRNA-transduced samples ...

... each serial dilution is plotted on the x-axis and its absorbance is plotted on the y-axis. With the help...spectrophotometer and concentration versus absorbance is plotted for the known concentrations (standard curve), ...concentration of a serially diluted standard is plotted against its OD560 measurements and the trendline...this kind of software, don’t give up! Rather than plotting the entire data set, plot only the linear portion...

...or siRNA expression from the other. The Q system Potter et al. from Liqun Luo’s lab developed the Q system...functional in C. elegans. Subsequent work by the Luo and Potter labs has refined the Q system and introduced the...Neurosci 9(5) (2006): 703-9. PubMed PMID: 16582903. Potter, Christopher J., et al. “The Q system: a repressible...publication at Addgene. Wei, Xing, Christopher J. Potter, Liqin Luo & Kang Shen. “Controlling gene expression...from this publication at Addgene. Riabinina O, Potter CJ. The Q-System: A Versatile Expression System...

...specifically how dry ice can affect virus from Addgene. Bottom line: there’s good news and there’s bad news. The...by a short 2-hour exposure to pH as low as 2.5 (Potter et al., 2014). They also showed that even 24 hours...infectivity after 3 weeks of exposure to pH 5.1 (Potter et al., 2014). Based on our results and the results...2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet K...

...-Linked Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides ...including but not limited to in situ hybridization, blotting (Northern and Southern) and affinity binding. ...PubMed PMID: 10470036. 3. Said, H. M. “Biotin: The forgotten vitamin.” American Journal of Clinical Nutrition...non-radioactive method for small RNA detection by northern blotting.” Rice 7.1 (2014): 1–7. PubMed PMID: 26224555....

...top strand first followed by the bottom strand versus nicking the bottom strand first followed by the top...PCR-generation of a mutant version of the top and bottom strands of the backbone plasmid. Unlike SDM, which... Does it matter what order I nick the top and bottom strands of the plasmid? The authors’ compared nicking...

...standard confocal microscopy. Image adapted from (Bottanelli et al., 2016) under CC-BY license. Combining...experiments! References and resources References Bottanelli, F., Kromann, E. B., Allgeyer, E. S., Erdmann...Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert...-39655-3 Wilhelm, J., Kühn, S., Tarnawski, M., Gotthard, G., Tünnermann, J., Tänzer, T., Karpenko, J.,...

...      *If you submitted the names Abi, Lin, or Blottie (our top 3 choices), we'll be in touch with you... three choices.   Fig. 1: Lin, Abi, or Blottie? Which one suits me best?     But between these... options, we simply can't decide! Abi, Lin, or Blottie? Which name best fits our new immunoglobulin friend...

...genome. (Bottom panel, right) A highly expressing RUBY plant is a vibrant red/magenta while (bottom panel...developing seeds from a homozygous RUBY plant. (Bottom panels) RUBY expression can vary from different...

...their RBE and T-shaped hairpin loop sequences (Wilmott et al. 2019). Most ITR sequences are from AAV2 ...cross-packaged with capsids from multiple AAV serotypes (Wilmott et al., 2019), and different combinations of ITR...doi.org/10.3389/fmed.2023.1106085. PMID: 36817775. Wilmott, P., Lisowski, L., Alexander, I. E., & Logan, G...

...has a less… distinctive smell. BME smells like rotting eggs but has a longer shelf life when stored properly...time; increased reagents Good luck and happy blotting! Many thanks to Addgenies Ashley Waldron, Meghan...blog Antibodies 101: The Basics of Western BlottingAntibodies 101: Selecting the Right AntibodyAntibodies...single protein controls in semi-quantitative immunoblotting. Journal of Neuroscience Methods, 172(2), 250...

...PVDF) membrane, which is sturdier and retains the blotted protein better than a nitrocellulose membrane. ...secondary antibody. This is the one time in western blotting that you’re looking for a “blank” result! If some...Resources on the Addgene blog The Basics of Western Blotting Find other tips and tricks for molecular biology...

..., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight...10.1021/acssynbio.8b00333 Pryor, J. M., Potapov, V., Bilotti, K., Pokhrel, N., & Lohman, G. J. S. (2022). Rapid....1c00525 Pryor, J. M., Potapov, V., Kucera, R. B., Bilotti, K., Cantor, E. J., & Lohman, G. J. S. (2020). ...

...and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al. 2016). That being..., parasites, and more (Bertiaux, et al. 2021; Gambarotto, et al. 2019; M’Saad, et al. 2020). Plus, it’...https://doi.org/10.1016/j.neuron.2010.11.021  Gambarotto D, Zwettler FU, Guennec ML, et al (2019) Imaging...