- Apr 2022
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twitter.com twitter.com
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Dr Ellie Murray, ScD. (2021, September 19). We really need follow-up effectiveness data on the J&J one shot vaccine, but not sure what this study tells us. A short epi 101 on case-control studies & why they’re hard to interpret. 🧵/n [Tweet]. @EpiEllie. https://twitter.com/EpiEllie/status/1439587659026993152
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www.covid-19-mobility.org www.covid-19-mobility.org
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Schlosser, F. (2021, November 29). Omicron—Import Risk. Covid-19 Mobility Project. https://www.covid-19-mobility.org/reports/importrisk_omicron/
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www.facetsjournal.com www.facetsjournal.com
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Caulfield, T., Bubela, T., Kimmelman, J., & Ravitsky, V. (2021). Let’s do better: Public representations of COVID-19 science. FACETS, 6, 403–423. https://doi.org/10.1139/facets-2021-0018
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www.ema.europa.eu www.ema.europa.eu
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EMA. (2020, October 27). COVID-19 vaccines: Development, evaluation, approval and monitoring [Text]. European Medicines Agency. https://www.ema.europa.eu/en/human-regulatory/overview/public-health-threats/coronavirus-disease-covid-19/treatments-vaccines/vaccines-covid-19/covid-19-vaccines-development-evaluation-approval-monitoring
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- Mar 2022
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twitter.com twitter.com
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ReconfigBehSci on Twitter: ‘@alexdefig are you really going to claim that responses to the introduction of passports on uptake across 4 other countries are evidentially entirely irrelevant to whether or not passports are justified or not?’ / Twitter. (n.d.). Retrieved 31 March 2022, from https://twitter.com/SciBeh/status/1444358068280565764
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www.science.org www.science.org
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Contents | Science 375, 6586. (n.d.). Science. Retrieved 23 March 2022, from https://www.science.org/doi/abs/10.1126/science.2022.375.issue-6586
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- Feb 2022
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Neue Studie: Wie gut schützen FFP2-Masken vor Corona? (2021, December 4). ZDFheute. https://www.zdf.de/uri/6f2f3223-fd82-4dc3-a6bf-24e8dc6eb648
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twitter.com twitter.com
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Deepti Gurdasani. (2022, January 29). Going to say this again because it’s important. Case-control studies to determine prevalence of long COVID are completely flawed science, but are often presented as being scientifically robust. This is not how we can define clinical syndromes or their prevalence! A thread. [Tweet]. @dgurdasani1. https://twitter.com/dgurdasani1/status/1487366920508694529
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twitter.com twitter.com
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Deepti Gurdasani. (2022, January 30). Have tried to now visually illustrate an earlier thread I wrote about why prevalence estimates based on comparisons of “any symptom” between infected cases, and matched controls will yield underestimates for long COVID. I’ve done a toy example below here, to show this 🧵 [Tweet]. @dgurdasani1. https://twitter.com/dgurdasani1/status/1487578265187405828
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- Jan 2022
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twitter.com twitter.com
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Retraction Watch. (2022, January 7). Our list of retracted COVID-19 papers is up to 206. For context and denominators, please see the post. Https://retractionwatch.com/retracted-coronavirus-covid-19-papers/ [Tweet]. @RetractionWatch. https://twitter.com/RetractionWatch/status/1479599196089077766
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- Dec 2021
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sciencebasedmedicine.org sciencebasedmedicine.org
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Loud Silenced Doctors | Science-Based Medicine. (2021, December 19). https://sciencebasedmedicine.org/muzzled/
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- Oct 2021
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twitter.com twitter.com
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@lakens: I’m interested to hear which COVID studies by psychologists you think have had a measurable positive effect on the world. E.g.… [Tweet]. https://twitter.com/lakens/status/1427856489990004741?s=20
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www.science.org www.science.org
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Mateus, J., Dan, J. M., Zhang, Z., Rydyznski Moderbacher, C., Lammers, M., Goodwin, B., Sette, A., Crotty, S., & Weiskopf, D. (n.d.). Low-dose mRNA-1273 COVID-19 vaccine generates durable memory enhanced by cross-reactive T cells. Science, 0(0), eabj9853. https://doi.org/10.1126/science.abj9853
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- Sep 2021
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www.theguardian.com www.theguardian.com
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The difficulties of estimating long Covid | David Spiegelhalter and Anthony Masters | The Guardian. (n.d.). Retrieved September 28, 2021, from https://www.theguardian.com/theobserver/commentisfree/2021/sep/26/the-difficulties-of-estimating-long-covid
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www.medrxiv.org www.medrxiv.org
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Aleta, A., Blas-Laína, J. L., Anglés, G. T., & Moreno, Y. (2021). Unraveling the COVID-19 hospitalization dynamics in Spain using publicly available data (p. 2021.09.03.21263086). https://www.medrxiv.org/content/10.1101/2021.09.03.21263086v1
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www.nature.com www.nature.com
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Bracher, J., Wolffram, D., Deuschel, J., Görgen, K., Ketterer, J. L., Ullrich, A., Abbott, S., Barbarossa, M. V., Bertsimas, D., Bhatia, S., Bodych, M., Bosse, N. I., Burgard, J. P., Castro, L., Fairchild, G., Fuhrmann, J., Funk, S., Gogolewski, K., Gu, Q., … Xu, F. T. (2021). A pre-registered short-term forecasting study of COVID-19 in Germany and Poland during the second wave. Nature Communications, 12(1), 5173. https://doi.org/10.1038/s41467-021-25207-0
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- Aug 2021
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www.nature.com www.nature.com
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Murphy, C., Laurence, E., & Allard, A. (2021). Deep learning of contagion dynamics on complex networks. Nature Communications, 12(1), 4720. https://doi.org/10.1038/s41467-021-24732-2
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- Jul 2021
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psyarxiv.com psyarxiv.com
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Inasaridze, K. (2021). Behavioral activation method for depression therapy [Preprint]. PsyArXiv. https://doi.org/10.31234/osf.io/ge8s3
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www.theguardian.com www.theguardian.com
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‘Trust the science’ is the mantra of the Covid crisis – but what about human fallibility? | Margaret Simons | The Guardian. (n.d.). Retrieved July 27, 2021, from https://www.theguardian.com/commentisfree/2021/jul/24/trust-the-science-is-the-mantra-of-the-covid-crisis-but-what-about-human-fallibility?CMP=Share_iOSApp_Other
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psyarxiv.com psyarxiv.com
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Park, A., & Velez, C. (2021). A mixed methods study of the psychological impact of the COVID-19 pandemic on American life. PsyArXiv. https://doi.org/10.31234/osf.io/tjz32
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- Jun 2021
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www.health.gov.au www.health.gov.au
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Health, A. G. D. of. (2021, March 13). COVID-19 vaccines – Is it true? [Text]. Australian Government Department of Health; Australian Government Department of Health. https://www.health.gov.au/initiatives-and-programs/covid-19-vaccines/is-it-true
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www.nature.com www.nature.com
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Derrick, G. (2021). A year after lockdowns began, has research got any kinder? Nature. https://doi.org/10.1038/d41586-021-01579-7
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www.theguardian.com www.theguardian.com
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WHO voices concerns over Sputnik V Covid vaccine plant. (2021, June 23). The Guardian. http://www.theguardian.com/society/2021/jun/23/who-voices-concerns-over-sputnik-v-covid-vaccine-plant
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- May 2021
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psyarxiv.com psyarxiv.com
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Agarwal, A. (2021). Ripple Effect of a Pandemic: Analysis of the Psychological Stress Landscape during COVID19. PsyArXiv. https://doi.org/10.31234/osf.io/dm5x2
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www.eventbrite.com www.eventbrite.com
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Data Collection and Integration to Enhance Public Health Registration, Thu, Jun 10, 2021 at 1:00 PM | Eventbrite. (n.d.). Retrieved May 28, 2021, from https://www.eventbrite.com/e/data-collection-and-integration-to-enhance-public-health-registration-156146370999
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psyarxiv.com psyarxiv.com
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Daly, M., & Robinson, E. (2020). Psychological distress and adaptation to the COVID-19 crisis in the United States [Preprint]. PsyArXiv. https://doi.org/10.31234/osf.io/79f5v
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www.mcgill.ca www.mcgill.ca
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Epidemiology 101 for Journalists. (n.d.). Epidemiology, Biostatistics and Occupational Health. Retrieved 23 February 2021, from https://www.mcgill.ca/epi-biostat-occh/epidemiology-101-journalists
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- Apr 2021
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www.thelancet.com www.thelancet.com
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Ten scientific reasons in support of airborne transmission of SARS-CoV-2—The Lancet. (n.d.). Retrieved April 19, 2021, from https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)00869-2/fulltext
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- Mar 2021
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www.cam.ac.uk www.cam.ac.uk
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Machine learning models for diagnosing COVID-19 are not yet suitable for clinical use. (2021, March 15). University of Cambridge. https://www.cam.ac.uk/research/news/machine-learning-models-for-diagnosing-covid-19-are-not-yet-suitable-for-clinical-use
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arxiv.org arxiv.org
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Gupta, Prateek, Tegan Maharaj, Martin Weiss, Nasim Rahaman, Hannah Alsdurf, Abhinav Sharma, Nanor Minoyan, et al. ‘COVI-AgentSim: An Agent-Based Model for Evaluating Methods of Digital Contact Tracing’. ArXiv:2010.16004 [Cs], 29 October 2020. http://arxiv.org/abs/2010.16004.
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edmontonjournal.com edmontonjournal.com
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Bergot, N. (2020, October 24). Battling fake science: University of Alberta launches free online science literacy course. Edmonton Journal. https://edmontonjournal.com/news/local-news/battling-fake-science-ualberta-launches-free-online-science-literacy-course
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www.collabovid.org www.collabovid.org
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Collabovid. (n.d.). Retrieved 6 March 2021, from https://www.collabovid.org/
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- Feb 2021
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www.cbc.ca www.cbc.ca
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Pennycook, Gordon. ‘How the COVID-19 Crisis Exposes Widespread Climate Change Hypocrisy’. CBC, 22 May 2020. https://www.cbc.ca/news/canada/saskatchewan/opinion-climate-change-should-believe-dont-understand-1.5482416.
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- Oct 2020
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twitter.com twitter.com
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Open Science Community Tilburg on Twitter. (n.d.). Twitter. Retrieved October 28, 2020, from https://twitter.com/OpenTilburg/status/1318518990000607234
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arxiv.org arxiv.org
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Brañas-Garza, P., Jorrat, D., Espín, A. M., & Sánchez, A. (2020). Paid and hypothetical time preferences are the same: Lab, field and online evidence. ArXiv:2010.09262 [Physics]. http://arxiv.org/abs/2010.09262
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www.youtube.com www.youtube.com
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Science as Amateur Software Development. (2020, September 26). https://www.youtube.com/watch?v=zwRdO9_GGhY&feature=youtu.be
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www.idunn.no www.idunn.no
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Krumsvik, R. J. (2020). Extended Editorial. Nordic Journal of Digital Literacy, 15(03), 141–152. https://doi.org/10.18261/issn.1891-943x-2020-03-01
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- Aug 2020
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covid-19.iza.org covid-19.iza.org
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Mitze, T., Kosfeld, R., Rode, J., & Wälde, K. (2020). Face Masks Considerably Reduce COVID-19 Cases in Germany: A Synthetic Control Method Approach. IZA Discussion Paper, 13319.
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www.biorxiv.org www.biorxiv.org
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Guo, L., Boocock, J., Tome, J. M., Chandrasekaran, S., Hilt, E. E., Zhang, Y., Sathe, L., Li, X., Luo, C., Kosuri, S., Shendure, J. A., Arboleda, V. A., Flint, J., Eskin, E., Garner, O. B., Yang, S., Bloom, J. S., Kruglyak, L., & Yin, Y. (2020). Rapid cost-effective viral genome sequencing by V-seq. BioRxiv, 2020.08.15.252510. https://doi.org/10.1101/2020.08.15.252510
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www.nber.org www.nber.org
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Baker, S. R., Bloom, N., Davis, S. J., Kost, K. J., Sammon, M. C., & Viratyosin, T. (2020). The Unprecedented Stock Market Impact of COVID-19 (Working Paper No. 26945; Working Paper Series). National Bureau of Economic Research. https://doi.org/10.3386/w26945
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van Rooij, I., & Baggio, G. (2020). Theory development requires an epistemological sea change [Preprint]. PsyArXiv. https://doi.org/10.31234/osf.io/rnw9q
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twitter.com twitter.com
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Travis Whitfill MPH on Twitter: “A quick visual aid of major studies & levels of evidence against #hydroxychloroquine for the use in COVID-19 patients. No robust studies have found any type of benefit for HCQ. https://t.co/YbSjvaoEoO” / Twitter. (n.d.). Twitter. Retrieved August 2, 2020, from https://twitter.com/twhitfill/status/1288825416975708161
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- Jul 2020
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www.nber.org www.nber.org
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Hortaçsu, A., Liu, J., & Schwieg, T. (2020). Estimating the Fraction of Unreported Infections in Epidemics with a Known Epicenter: An Application to COVID-19 (Working Paper No. 27028; Working Paper Series). National Bureau of Economic Research. https://doi.org/10.3386/w27028
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osf.io osf.io
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Dudel, C., Riffe, T., Acosta, E., van Raalte, A. A., Strozza, C., & Myrskylä, M. (2020). Monitoring trends and differences in COVID-19 case fatality rates using decomposition methods: Contributions of age structure and age-specific fatality [Preprint]. SocArXiv. https://doi.org/10.31235/osf.io/j4a3d
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osf.io osf.io
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Weed, M. (2020). Models and methods to analyse the interaction of evidence and policy in the first 100 days of the UK government’s response to COVID-19 (v1.1). https://doi.org/10.31235/osf.io/f73u4
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psyarxiv.com psyarxiv.com
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Fuhrer, J., & Cova, F. (2020). “Quick and dirty”: Intuitive cognitive style predicts trust in Didier Raoult and his hydroxychloroquine-based treatment against COVID-19. https://doi.org/10.31234/osf.io/ju62p
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- Jun 2020
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www.theguardian.com www.theguardian.com
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'We use Shakespeare's heartbeat': lockdown theatre for autistic audiences.(n.d.). The Guardian. Retrieved June 17, 2020 from https://www.theguardian.com/stage/2020/jun/17/shakespeare-autistic-audiences-lockdown-flute-theatre-pericles
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www.iza.org www.iza.org
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Mitze, T., Kosfeld, R., Rode, J., & Wälde, K. (2020). Face Masks Considerably Reduce COVID-19 Cases in Germany: A Synthetic Control Method Approach (No. 13319). Institute of Labor Economics (IZA).
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Horn, S. R., Weston, S. J., & Fisher, P. (2020). Identifying causal role of COVID-19 in immunopsychiatry models [Preprint]. PsyArXiv. https://doi.org/10.31234/osf.io/w4d5u
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- May 2020
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Mei, X., Lee, H.-C., Diao, K., Huang, M., Lin, B., Liu, C., Xie, Z., Ma, Y., Robson, P. M., Chung, M., Bernheim, A., Mani, V., Calcagno, C., Li, K., Li, S., Shan, H., Lv, J., Zhao, T., Xia, J., … Yang, Y. (2020). Artificial intelligence for rapid identification of the coronavirus disease 2019 (COVID-19). MedRxiv, 2020.04.12.20062661. https://doi.org/10.1101/2020.04.12.20062661
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www.preprints.org www.preprints.org
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Samuel, J.; Ali, G.G.M.N.; Rahman, M.M.; Esawi, E.; Samuel, Y. COVID-19 Public Sentiment Insights and Machine Learning for Tweets Classification. Preprints 2020, 2020050015 (doi: 10.20944/preprints202005.0015.v1)
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www.nature.com www.nature.com
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West, R., Michie, S., Rubin, G. J., & Amlôt, R. (2020). Applying principles of behaviour change to reduce SARS-CoV-2 transmission. Nature Human Behaviour, 1–9. https://doi.org/10.1038/s41562-020-0887-9
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www.medrxiv.org www.medrxiv.org
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Modi, C., Boehm, V., Ferraro, S., Stein, G., & Seljak, U. (2020). Total COVID-19 Mortality in Italy: Excess Mortality and Age Dependence through Time-Series Analysis. MedRxiv, 2020.04.15.20067074. https://doi.org/10.1101/2020.04.15.20067074
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- May 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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The CLD-J domain shares ~51 % similarity with the CDPK from Arabidopsis thaliana AtCPK-l. The homology model of CLD-JD was determined using Swiss Model from EMBL. The template model used was CLD-JD of AtCPK-1, which was crystallized as a dimer. The J -domain helices from the two monomers were swapped with each other in this structure (Chandran et aI., 2006). Therefore, the initial homology model generated for the complementary CLD-J domain for PfCDPK4 was also a dimer. To understand the interaction of this helix (Gln358_ Lys371) with CLP of the monomer, this helix was rotated and translated keeping residues 372-375 as the flexible linker region and superimposed on to the helix from the other monomer, which resulted in the initial model for the CLD-J domain monomer. Initially, these flexible linker residues (372-375) were locally minimized using COOT (Emsley and Cowtan, 2004), and the overall structure was refined with slow cooling using annealing of CNS (Brunger et aI., 1998) to remove all the short contacts. Finally, the model quality was checked with the Pro check software (Laskowski et aI., 1996). The homology model was generated with the help of Dr. S. Gaurinath, JNU
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Homology Modeling
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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released from the para-ovarian pad of fat as well as from the peritoneal reflections while care was taken to avoid injury to the ovarian vessels. A ligature was tied around the distal end of the fallopian tube including the ovarian vessels following which the ovary was excised. Hemostasis was secured before the stump of the tube was pushed back into the peritoneal cavity. The peritoneum was closed by continuous sutures using 2-0 silk. The same protocol was followed to perform oophorectomy on the contralateral side. The muscular layer and skin were closed together using surgical clips
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The anesthetized mice were operated under strict aseptic conditions inside a laminar flow hood. The mouse was placed over layers of sterile tissue paper and the skin overlying the dorsal flanks was sterilized by wiping with 70% ethanol. The flank was palpated gently to identify the kidney, and an incision(~ 5 mm) was made using a pair of scissors on the overlying skin which penetrated the skin, sub-cutaneous tissue and the muscle layer with the parietal peritoneum being exposed and intact. The para-ovarian pad of fat was identified through the intact peritoneum and a small incision was made on the peritoneum overlying it. The ovarian tissue along with the fallopian tube was mobilized and delivered through the incision site. The ovary was
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Bilateral oophorectomy
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Hair from the skin overlying the left and right dorsal flanks were removed using electrically operated razor. The skin overlying the abdomen was sterilized by wiping with 70% ethanol. Ketamine (1 00 mg/kg) and xylocaine (2%) (20 mg/kg) were mixed and administered intraperitoneally. The mice were returned to the cage and the onset of anesthetic effect was monitored. The mice were considered to be in surgical anesthesia when there was loss of palpebral reflex, righting reflex, and toe pinch reflex. Respiratory rate and heart rate were monitored continuously.
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General anesthesia:
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Bilateral oophorectomy, the surgical removal of both the ovaries, was performed in mice to simulate a condition of estrogen depletion. All procedures in mice were performed after obtaining approval from the Institutional Animal Ethics Committee (National Institute of Immunology, New Delhi). Female BALB/c mice were used in the study
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Bilateral oophorectomy and sham surgery in mice
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Animal experiments
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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in IOmM Tris/HCI (pH 7.4)/ lmM EDTN 0.5% SDS. The radioactivity associated with the cells was counted in a y-counter (LKB).
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Competition binding analysis was performed to compare the affinity of the chimeric toxins with native antibody. Anti-transferrin receptor antibody (HB21) was iodinated using iodogen method as described by Harlow and Lane, (1988). Adherent cell lines, namely, A431 and A549 were plated at 4Xl05 cells per well in a 24 well plate and used 16 h later for the assay. 500 J.Ll medium, same as described in cytotoxicity assay, was used to dispense the cells. HUT102 cells were also plated at the same density in microfuge tubes and used immediately. After 2 washes with binding buffer (0.1% BSA in DMEM), various dilutions of toxin, along with 3 ng of labeled antibody in binding buffer, were added to the cells. The cells were incubated at 25 °C for 2 h with mild shaking, washed three times with binding buffer and lysed
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Binding Studies
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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then estimated by monitoring its competitive binding to the hCG recepto~ in the presence of radiolabelled hCG. The testes homogenates from 10 -14 week old Wistar outbred rats were prepared by the method of Dighe and Moudgal ( 1983 ), and the receptor assay 1975 ), with 1989 ) .
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Culture supernate from stable clones secreting alpha hCG was mixed with the culture supernate from stable clones showing J3hCG activity. The mixing was done with shaking at 37°C for 16 hours, to allow the two subunits to associate. The presence of the heterodimer in the culture supernate was
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Radioreceptor assay.
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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EROD
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ITALYusinga96wellmicrolitreplatereadbyELISAmicroplatereader(modelEL3/Sx,BioTeKInstrumentsINC).Thefinalsolutionwasreadatawavelengthof450nm.Theplasmacortisolconcentrationwascalculatedbasedonaseriesofstandards.
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PlasmacortisolwasmeasuredbyadirectimmunoenzymaticdeterminationofcortisolkitmanufacturedbyEquiparSriviaG.Ferrari,21/N-21047,SARONNO
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Plasmacortisol
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Thecapabilityofheadkidneyneutrophilstomovewasassayedbyamigration-under-agarosetechniquemodifiedfromNelsonetal.(1975).ThemethodhasbeendescribedbySaloetal.(1998).Thedistance,thecellshadmigratedfromthemarginofthewelltowardsthewellcontainingcasein(directedmigration)andintheopposite direction(randommigration)weremeasuredunderthemicroscope.
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Migration
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Phagocytesfromtheheadkidneywerestimulatedwithphorbol12-myristate13 -acetate(PMA,SigmachemicalCo)andtherespiratoryburstwasdeterminedbytheluminol-enhancedCLmethod(ScottandKlesius,1981)
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Theenzyme-linkedimmunospot(ELISPOT)assaywasusedfortheenumeration of totalimmunoglobulinsecretingcells(ISC)andantigen(BCG)-specificantibody-secretingcells(ASC)inthespleenandtheblood.TheELISPOTassayfollowedby Aaltonenetal.(1994)wasused.
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EnumerationofsecretinglymphocyteswithELISPOTassay
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TheamountoftotalIgMandspecificantibovineY-globulin(BGG)antibodyinthefishplasmawasmeasuredbyanenzyme-linkedimmunosorbentassay(ELISA)asgiven byAaltonenetal.(1994)
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Serumimmunoglobulin
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Theheadkidneywasusedasasourceofphagocytesforchemiluminescence (CL)andmigrationassays.Cellsfromthehomogenizedheadkidneywereseparatedwithatwo-stepPercollgradient.Granulocyteswerecollectedatthe1.070-1.090g/cm3interfaceandafterwashingwithrHBSSresuspendedinphenolred-freerRPMl.
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Headkidney
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Thespleenswereremovedandhomogenisedindividuallythroughanylonnet(80mesh).Forisolationoflymphocytes,thetissuehomogenatewaslayeredonatwo-stepPercolldensitygradientandcentrifugedfor30minat400xg.Thelymphocyteswerecollectedatthe1.040-1.080g/cm3interface,washedtwice(400xg,10min)withrHbss,andresuspendedin2mlrRPMl.Cellswerecountedbytrypanblueexclusioninahaemocytometer(viability>95%)andthenumberoflymphocytesfrombloodandspleenwereadjustedto2x106/ml
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Spleen
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Abloodsamplewastakenfromthecaudalveinofeachfishwitha1mlheparinisedsyringeand24-gaugeneedle.Thebloodwascentrifuged(400xg,5min)fortheseparationofplasma.Plasmawasstoredfrozen(-70°C)forthedeterminationoftheimmunologicalparameters
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Collectionofplasma
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Immunologicalanalysis
Tags
- Method-19-Method-7
- Method-19-Method-5-detail
- Method-19-Method-4-detail
- Method-19-Method-1
- Method-19-Method-2-detail
- Method-19
- Method-19-Method-9
- Method-19-Method-4
- Method-19-Method-1-detail
- Method-19-Method-5
- Method-19-Method-2
- Method-19-Method-8
- Method-19-Method-6
- Method-19-Method-7-detail
- Method-19-Method-8-detail
- Method-19-Method-3
- Method-19-Method-3-detail
Annotators
URL
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Results are expressed as mean ±SE. An unpaired two tailed student's t-test using Sigma Plot Version 10 & 11 was used for statistical analys
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Statistical analysis
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and incubated at RT for 5 min. The O.D. was measured at 595nm spectrophotometrically and quantitated against a standard reference table supplied with the kit. Alternatively, a standard curve was also plotted using different concentrations of Bovine Serum Albumin (BSA) and used to quantitate protein concentration. Electrophoretic separation of proteins: Electrophoresis for protein separation was performed using the Laemmli buffer system (Laemmli, 1970) on 10 or 12% polyacrylamide gels under reducing or denaturing conditions (SDS-PAGE). Polyacrylamide gels were prepared from 30% acrylamide (30% acrylamide, 0.8% bis-acrylamide), 4X running buffer (1.5M Tris-HCl pH 8.8), 4X stacking buffer (O.SM Tris-HCl pH 6.8), 10% SDS and 10% ammonium persulfate (APS). The gels were run in tank buffer (0.025M Tris Base, 0.192M glycine and 0.1% w I v SDS) at 40-60V while samples were in stacking gel and 80-100V once they entered resolving gel. A molecular weight marker (unstained or pre-stained depending on the requirement) containing proteins of known sizes were run to evaluate the approximate molecular weights of the resolved proteins. Visualization of protein bands on Polyacrylamide gels: The protein bands resolved in the gels were stained with Coomassie Brilliant Blue R250 (0.125%w lv CBB R250, 50% v lv methanol, 10% v lv acetic acid) for 15-30 min, followed by washing off excess stain with destain (50%v lv methanol, 10%v lv acetic acid)
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SDS-PAGE is used to separate proteins according to their electrophoretic mobility which is a function of the length of the polypeptide chain or molecular weight, in the presence of denaturating agents when the secondary structure is lost. One molecule of SDS binds every 2 amino acids, imparting a net negative charge that is proportional to the length of the polypeptide. When loaded on the gel matrix and placed under an electric field, the negatively charged proteins migrate towards the positively charged electrode and are separated by molecular sieve. Preparation of lysates: After appropriate treatments, cells were harvested and washed 1X in PBS to remove any traces of medium and FBS. The remaining pellet was resuspended in residual buffer. A minimal volume of 2X sample buffer (0.125M Tris HCI pH 6.8, 4% sodium dodecyl sulphate, 20%v /v glycerol, 10% P-mercaptoethanol and 0.01 % bromo-phenol blue) was added drop-wise to the pellet while vortexing to ensure complete lysis. The lysate was denatured by boiling at 99°C for 15 min and debris pelleted down by centrifugation at 10000 x g for 5 min at RT. The supernatant was decanted into a fresh tube and used immediately or stored at -70°C. Estimation of protein concentration in lysates: Total protein concentration of whole celllysates was performed using the CB-X™ Protein Assay kit, by a modification of the Bradford method (Bradford, 1976). In brief, 1mL of chilled (-20°C) precipitant solution was added to 10pL of the whole cell lysate and centrifuged at 10,000 x g for 10 min. The supernatant was discarded and the pellet resuspended in 50pL each of solubilising solution A and B. To this 1mL colour reagent was added, the samples were vortexed
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Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis
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