Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.

It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads

Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.

Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

BTW, like to have information on the spin column kit? Here it is: http://www.allelebiotech.com/gfp-nab-agarose-spin-kit-20-reactions/

Tags: agarose beads, camelid antibody, capture reagent, co-IP 101, GFP, gfp affinity tag, gfp antibody, GFP co-IP, GFP-nAb, magnetic beads, mNeonGreen-nAb, nano antibodies, nanobodies, polyacrylamide beads, RFP, spin column, Trap, VHH domain

Record number of papers citing the GFP-Trap group products in 2011

The following are references in regards to GFP Trap published in the second half of 2011 (not a complete list); a high quality GFP-binding protein based on a single domain antibody derived from Camelids. It is characterized by a small barrel shaped structure (13 KDa, 2.5nm X 4.5 nm) and a very high stability (stable up to 70°C, functional within 2M NaCl or 0.5% SDS). With much greater stability, specificity, and affnity, GFP-Trap®, the recent addition to antibodies for immunoprecipitation, should make GFP the most suitable tag for immunoprecipitation assays.

For live PubMed links, view this version please.

Krastev, D. B., Slabicki, M., et al. (2011). A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly. Nature Cell Biology. 13: 809-818. PubMed

Aboobakar, E. F., Wang, X., et al. (2011). The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high temperature stress responses in Cryptococcus neoformans. Eukaryotic Cell. EC. 05148-05111v05141. PubMed

Uhrig, R. G. and Moorhead, G. B. G. (2011). Two ancient bacterial-like PPP family phosphatases from Arabidopsis thaliana are highly conserved plant proteins that possess unique properties. Plant Physiology. PubMed

Larance, M., Kirkwood, K. J., et al. (2011). Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway. Molecular & Cellular Proteomics. PubMed

Hattersley, N., Shen, L., et al. (2011). The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Molecular Biology of the Cell. 22: 78-90. PubMed

Rancz, E. A., Franks, K. M., et al. (2011). Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics. Nature Neuroscience. 14: 527-532. PubMed

Palmer, C. S., Osellame, L. D., et al. (2011). MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO reports. 12: 565-573. PubMed

Pichler, G., Wolf, P., et al. (2011). Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways. Journal of Cellular Biochemistry. 112: 2585-2593. PubMed

Mitchell, L., Lau, A., et al. (2011). Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4. PLoS One. 6: e25336. PubMed

Engeland, C. E., Oberwinkler, H., et al. (2011). The cellular protein Lyric interacts with HIV-1 Gag. Journal of virology. JVI. 00174-00111v00171. PubMed

Wang, C. and Youle, R. (2011). Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak. Oncogene. PubMed

Tulloch, L. B., Howie, J., et al. (2011). The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation. Journal of Biological Chemistry. 286: 36020-36031. PubMed

Sun, L. and Wang, C. C. (2011). The Structural Basis of Localizing Polo-Like Kinase to the Flagellum Attachment Zone in Trypanosoma brucei. PLoS One. 6: e27303. PubMed

Bouttier, M., Saumet, A., et al. (2011). Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation. Nucleic acids research. PubMed

Matos, J., Blanco, M. G., et al. (2011). Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis. Cell. 147: 158-172. PubMed

Nagel, C. H., Albrecht, N., et al. (2011). Herpes Simplex Virus Immediate-Early Protein ICP0 Is Targeted by SIAH-1 for Proteasomal Degradation. Journal of virology. 85: 7644. PubMed

Studencka, M., Konzer, A., et al. (2011). Novel roles of C. elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression. Molecular and Cellular Biology.PubMed

Muehlen, S., Ruchaud-Sparagano, M. H., et al. (2011). Proteasome-independent Degradation of Canonical NFŒ?B Complex Components by the NleC Protein of Pathogenic Escherichia coli. Journal of Biological Chemistry. 286: 5100. PubMed

Galan, J. A., Paris, L. L., et al. (2011). Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap. Journal of the American Society for Mass Spectrometry. 1-10. PubMed

Chamousset, D., De Wever, V., et al. (2010). RRP1B Targets PP1 to Mammalian Cell Nucleoli and is Associated with Pre-60S Ribosomal Subunits. Mol Biol Cell. PubMed

Kovacs, E. M., Verma, S., et al. (2011). N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nature Cell Biology. 13: 934-943. PubMed

Boysen, K. E. and Matuschewski, K. (2011). Arrested oocyst maturation in Plasmodium parasites lacking type II NADH: ubiquinone dehydrogenase. Journal of Biological Chemistry. 286: 32661-32671. PubMed

Mortusewicz, O., Fouquerel, E., et al. (2011). PARG is recruited to DNA damage sites through poly (ADP-ribose)-and PCNA-dependent mechanisms. Nucleic acids research. 39: 5045. PubMed

Graewe, S., Rankin, K. E., et al. (2011). Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathogens. 7: e1002224. PubMed

Yang, X. D., Huang, S., et al. (2011). Distinct and mutually inhibitory binding by two divergent Œ?-catenins coordinates TCF levels and activity in C. elegans. Development. 138: 4255-4265. PubMed

Pollithy, A., Romer, T., et al. (2011). Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense. Applied and environmental microbiology. 77: 6165-6171. PubMed

Kozubowski, L., Thompson, J. W., et al. (2011). Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics. PLoS One. 6: e25280. PubMed

Garcia-Gomez, J. J., Lebaron, S., et al. (2011). Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae. Molecular and Cellular Biology. 31: 4156-4164. PubMed

Van Damme, D., Gadeyne, A., et al. (2011). Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proceedings of the National Academy of Sciences. 108: 615. PubMed

Qvist, P., Huertas, P., et al. (2011). CtIP Mutations Cause Seckel and Jawad Syndromes. PLoS Genetics. 7: e1002310. PubMed

Labella, S., Woglar, A., et al. (2011). Polo Kinases Establish Links between Meiotic Chromosomes and Cytoskeletal Forces Essential for Homolog Pairing. Developmental Cell. PubMed

Harterink, M., Port, F., et al. (2011). A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biology. 13: 914-923. PubMed

Konopacki, F. A., Jaafari, N., et al. (2011). Agonist-induced PKC phosphorylation regulates GluK2 SUMOylation and kainate receptor endocytosis. Proceedings of the National Academy of Sciences.PubMed

Chuhma, N., Tanaka, K. F., et al. (2011). Functional connectome of the striatal medium spiny neuron. The Journal of Neuroscience. 31: 1183-1192. PubMed

Jackson, B. R., Boyne, J. R., et al. (2011). An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export. PLoS Pathogens. 7: e1002138. PubMed

Tags: anti-GFP, camelid antibodies, Chromotek, Co-IP, co-IP 101, GFP-Trap, GFPTrap, immunoprecipitation, nAb: Camelid Antibodies, nanobodies, pulldown, RFPTrap, VHH, VHH antibody