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

The Development of mNeonGreen

This week our most recent publication, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum” will be published in Nature Methods. It has already been viewable online for some time now, here is a link. We believe this new protein possesses a great deal of potential to advance the imaging fields through enhanced fluorescent microscopy. mNeonGreen enables numerous super resolution imaging techniques and allows for greater clarity and insight into one’s research. As a result of this we are taking a new approach at Allele for distribution of this protein, and here we will describe the history of the protein and some of the factors that led us down this path.

mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology and the Scintillon Institute through the directed evolution of a yellow fluorescent protein we offer called LanYFP. LanYFP is a super bright yellow fluorescent protein derived from the Lancelet fish species, characterized by its very high quantum yield, however, in its native state LanYFP is tetrameric. Dr. Shaner was able to monomerize the protein and enhance a number of beneficial properties such as photostability and maturation time. The result is a protein that performs very well in a number of applications, but is also backwards compatible with and equipment for GFP imaging.

Upon publication there was a question of how distribution should be structured. How would we make this protein available to researchers in a simple manner was a very difficult challenge? We also relied heavily on Dr. Shaner’s knowledge and experience in these matters, as he related his experiences to us from his time in Roger Tsien’s lab at UCSD. When the mFruits was published their lab was inundated with requests. The average waiting period was 3 months to receive a protein and they required a dedicated research technician to handle this process. Eventually the mFruits from the Tsien lab were almost exclusively offered through Clontech. Thus we decided that Allele Biotechnology would handle the protein distribution and take a commercial approach to drastically decrease the turnaround time. The next challenge we faced was how to charge for this protein. Due to the cost of developing this protein, which was fully funded by Allele, there is a necessity to recoup our investment and ideally justify further development of research tools, but we also understand the budget constraints every lab now faces. From this line of thinking we conceived our group licensing model; we wanted to limit the charge to $100 per lab. The way this is fiscally justifiable is having every lab in a department or site license the protein at this charge, including access to all related plasmids made by us as well as those generated by other licensed users (Click here for our licensing page). The benefit we see to this is that the protein is licensed for full use at a low cost, and collaboration amongst one’s colleagues is not only permissible, it’s encouraged. We saw this as a win-win situation. We would recoup our cost and invest in further fluorescent protein research, and our protein costs would not be a barrier to research and innovation.

The granting of a license to use but not distribute material is not unique to commercial sources. Although academic material transfer agreements typically contain specific language forbidding distribution of received material beyond the recipient laboratory, some researchers choose to disregard these provisions. Unfortunately through this action they are disrespecting the intellectual property rights of the original researchers as well as violating the terms of the legal contract they signed in order to receive the material. We believe most researchers choose to respect the great deal of effort that goes into the creation of research tools for biology and do not distribute any material received from other labs without their express permission. However for a company that funds its own basic research our focus is often on the former example rather than the latter. We believe that this focus artificially drives up the costs of licensing a fluorescent protein and obtaining the plasmid, thus we have chosen to believe researchers will respect our intellectual property as long as we are reasonable in our distribution which is something we have truly striven for.

Additionally we believe the broad-range usage of a superior, new generation FP is an opportunity to advocate newer technologies that can be enabled by mNeonGreen, together with a number of Allele’s other fluorescent proteins (such as the photoconvertible mClavGR2, and mMaple). These new imaging technologies are called super resolution imaging (MRI). They provide researchers with a much finer resolution of cellular structures, protein molecule localizations, and protein-protein interaction information. We have started the construction of a dedicated webpage to provide early adopters with practical and simple guidance, click here to visit our super resolution imaging portal.

Tags: cell imaging, fluorescent protein, FP, GFP, mNeonGreen, Superresolution

Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

Ain't they pretty?

These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.

Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.

The mWasabi is stimulating

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

The LanFPs express well in bacteria.

The LanFPs express well in bacteria

Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.

The FPs fold so strongly that they fluorescence even in SDS-PAGE.

Can you see the FP bands in the SDS PAGE?

FPs in SDS PAGE–a closer look

Can you see them now?

FPs in gel cassette over UV lights

Invincible FPs

FPs in gel cassette under blue LED

Fluorescence in SDS page under blue LED

The purified FPs can be used as “real time” protein markers.

New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.

Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726

Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures

17 Papers Using GFP-Trap, 12 Since 2009

1. MacKay C, Déclais AC, Lundin C et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142:65-76.

2. Babiano R, de la Cruz J. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 2010 Apr 14.

3. Fulcher AJ, Dias MM, Jans DA. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem. 285:17744-53.

4. Taniue K, Nishida A, Hamada F et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development. 137:1755-64.

5. Rottach A, Frauer C, Pichler G et al. (2010). The multi-domain protein Np95 connects DNA methylation and histone modification. Nucleic Acids Res. 38:1796-804.

6. Boulon S, Ahmad Y, Trinkle-Mulcahy L et al. (2010). Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9:861-79.

7. Schornack S, Fuchs R, Huitema E et al. (2009). Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60:744-54.

8. Fellinger K, Bultmann S, Rothbauer U et al. (2009). Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells. EMBO Rep. 10:1259-64.

9. Muñoz IM, Hain K, Déclais AC et al. (2009). Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol Cell. 35:116-27.

10. Webby CJ, Wolf A, et al. (2009). Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing. Science. 325:90-93.

11. Rogowski K et al. (2009). Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell. 137: 1076-87.

12. Frauer C, Leonhardt H, (2009) A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding. Nucleic Acid Res. 35: 5402-5409.

13. Trinkle-Mulcahy L et al., (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183: s223-39.

14. Rothbauer U, Leonhardt H, (2008) Connecting Biochemistry and Cell Biology with Nanobodies. Zellbiologie aktuell 34: 9-12.

15. Rothbauer U et al., (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282-289.

16. Agarwal N et al., (2007) MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. Nucleic Acid Res.35: 5402-5409.

17. Rothbauer U et al., (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3: 887-889.

New Product of the Week 071910-072510: Cre Reporter Cell Line: LoxP-RFP Human Fibroblast, perfect to test our Cre-2A-GFP lentivirus, when cre works, the cell change from red to green.

Promotion of the Week 071910-072510: High Quality dNTP Mix, 10mM, 5 ml, $409 this week $309. Hurry, email to oligo@allelebiotech.com or fax 858-587-6692 by Sunday to save $100 on your lab budget.

Tags: anti-GFP, chromatin immunoprecipitation (ChIP), coIP, GFP, immunoprecipitation, nAb: Camelid Antibodies, Nanobodies, VHH, pull down

Lentivirus-lanYFP Give Away

Promotion of the week 03-22-10 to 03-28-10: To help researchers get familiar with pre-packaged lentivirus, we offer free high titer lentivirus carrying a truly bright and fast maturing lanYFP (lancelet FP, new exclusively from Allele).  Infect virtually any mammalian cells by a single manipulation (pipeting) and watch cells turn green/yellow in about a day under microscope or on FACS.  Primarily a yellow FP, lanYFP will show brighter fluorescence than EGFP even when observed using standard GFP/FITC filter set.

New product of the week 03-22-10 to 03-28-10: anti-mTFP1/mWasabi polyclonal antibodies.  It is tailored-made for Alleleustrious mTFP1 and mWasabi, the brightest teal and green FPs.

Tags: EGFP, Fluorescent proteins, GFP, Lancelet, lentivirus, mTFP1, mWasabi, pre-validated virus, YFP