About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011

With their ability to quantitatively pulldown GFP-tagged proteins, GFP-Trap (or RFP-Trap for DsRed-derived fluorescent proteins) beads have gained ground in becoming the reagent of choice for immuno-coprecipitation. The complexes isolated from GFP-Trap agarose or magnetic beads can be easily analyzed without interference from light or heavy IgG chains typically present after monoclonal or polyclonal antibody precipitation. Since the market launch of GFP-Trap, in each of the past 3 years, the number of publications citing GFP-Trap more has than doubled and there is no sign of that rate slowing down any time soon.

In 2011 alone, 48 research groups have published their results with data generated through use of GFP-Trap (not including other related products such as GFP-Booster, GFP-MultiTrap). Research topics in these recent publications include identification of domains of the zinc finger protein 638 (ZNF638) that interacts with C/EBP? when promoting adipocyte differentiation [1]; identification of phosphorylation site on Cdc42-associated kinase (Ack) by LC-MS/MS after immunoprecipitation [2]; and analysis of the activities of myosin heavy-chain kinases (MHCKs) in wild-type vs Htt mutant Dictyostelium discoideum, a cellular model for studying the Huntingon disease [3].

The use of GFP-Trap beads is a simple bind-wash-elute procedure that involves just one antibody already immobilized on either agarose or magnetic beads. Camelid antibodies, especially their VHH single domain fragments such as those used in GFP-Trap or RFP-Trap, are very stable (they can be shipped and temporarily stored at room temperature). The consistency of performance is very high; as a matter of fact, this line of products requires the lowest amount of technical support among all of our products. If you are still using tags like FLAG, V5, HA, etc., you should consider trying GFP as both a fluorescence and co-IP tag in your future experiments for obtaining results you previously could not obtain.

New Product of the Week: Non-Integrating iPSC Generation Kits. First of its kind on the market. Click to read more about mRNA-based reprogramming.

Promotion of the Week: Save 15% to save the environment by using EcoCulture Dishes at 30% less plastic for better imaging. Code: 091911DISH when call or email us.

Blog References:
[1] Meruvu, S. et al. “Regulation of Adipocyte Differentiation by the Zinc Finger Protein ZNF638″ JBC 2011
[2] Shen, H. et al. “Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin” Molecular Biology of the Cell 2011
[3] Wang, Y. et al. “Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation” Molecular Biology of the Cell 2011

北岛以一句“卑鄙是卑鄙者的通行证，高尚是高尚者的墓志铭”而著名

"今天他当然更清楚，在一个不要墓志铭的世界，卑鄙者的通行证是抢破头的。所以他也迫不及待地实践自己的名言去了。" 曹长青：北岛回国跟宣传部长唱红诗


 可叹，又一个张艺谋。其实为求名逐利也不一定是这些“精英”表现人格低下的原因，很多时候他们只是看不见或者没时间去看世上的邪恶与不公正。

这两年我有机会与之交谈世事的北大同学中，除一人有共鸣，一人观点相反并激烈辩论外，多数的反应是问我是否CND看多了，或者是不是FLG。与我辩论者的言辞基本符合网上所谓“五毛”的观点，只是更自信一些，让我觉得很多被人在网上贴“五毛”标签的人可能不是拿钱发贴，而确实相信那些别人不相信他们会相信的观点。不管你信不信，反正我相信我的在美国读过博士，现在美国工作，多半是美国公民的大学同学不是“五毛”。人的观点的差距可以是巨大的，即使从表面上看来我们有着相似的背景。

另一同学曾道:"我已经可以退休了，你还在奋斗。。。“ Yeah right! 就象你一辈子的求学、训练、竞争、工作都只是为了尽快走到退休那一步。如果只为自己或自己的孩子们想，要退休当然可以，在自己的庄园上在种些果树养些动物，钓鱼打猎都是乐趣，或者写一些书，文学、历史、科学史亦无不可。但正像Richard Gere  在 Primo Fear 里说的”If you can still play the game (be a trial lawyer), why become a referee (court judge)?" I am still in my prime for science, and fight for what I believe is right.

Large Intergenic Non-Coding RNAs (lincRNAs) Play Large Roles in Pluripotency

AlleleNews

here may be only 21,000 distinct proteins encoded in a human genome but there are also thousands of transcripts that do not encode any proteins in human cells.  These so called lincRNAs have been a subject of speculations in terms of functions and mechanisms underlying the functions.  In a recent Nature Article, Eric Lander’s group at Harvard and their colleagues have shown by RNAi that ES-cell expressed lincRNAs function in keeping pluripotency.  They seem to maintain expression of pluripotency-related factors such as Oct4 and Nanog, while at the same time lincRNA genes appear to be targets of these factors’ transcriptional control.

As shown by immuneprecipitation, lincRNAs bind to chromatin proteins such as those of the polycomb family and influence chromatin structures and gene expression, at least as part of their functional mechanism.  It would be interesting if the RNAi-against-lincRNAs experiments were complemented by transgene expression, something we might see in follow-up mechanism studies on lincRNAs.  Pulldown of lincRNAs instead of the polycomb proteins could be helpful in identifying their unknown partners. 

It will also be interesting to see how lincRNA profiles correlate with iPSCs created by different methods or from different sources.  It may be worth it to even try and use lincRNAs as reprogramming reagents as mi-RNA302, miRNA369, etc.

Guttman et al.  2011, Nature

http://news.allelebiotech.com/index.php?mod=article&cat=iPSStemCells&article=1841 

Methods of iPSC Generation Update

AlleleBlog

Induced pluripotent stem cells can be directly generated from adult cell cultures through the introduction of a group of factors, e.g. Oct4, Sox2, Klf4, and c-Myc (the Yamanaka factors) [Takahashi and Yamanaka, 2006]. Additional factors such as Nanog and Lin28 can either substitute some of the Yamanaka factors or supplement them for higher reprogramming efficiency [Yu et al. 2007].

The original pluripotent stem cells induction methods involved retrovirus or lentivirus that would leave foot-print in the host genome, a concern for clinical use of iPSCs. Several groups have tried to create iPSCs without integrating viruses, such as using small molecules, directly delivering proteins instead of cDNAs, viruses with RNA genomes, episomal systems, or removable elements such as PiggyBac or Sleeping Beauty transposons. From the literature and our first-hand experience in the iPS market, none of these methods has become a widely applicable tool, mostly due to impractically low reprogramming efficiency.

In addition to low efficiency, RNA viruses, such as the sendai virus, are still viruses and have virus-associated risks. Episomal plasmids or removable transposons still involve DNA, so the possibility of genomic integration by recombination remains. In case of some transposons such as PiggyBac, there is an additional question about the degree of removal – whether it is certain that all integrated transposons, often inserted within genes, are deleted; in case of transposons similar to Sleeping Beauty, the small footprints they leave behind may post a concern.

The method of choice for generating zero-footprint iPSCs should clearly be RNA-based without the involvement of virus. Luigi Warren and his former colleagues at Harvard demonstrated that by using in vitro transcribed iPS factor mRNAs with modified CTP and UTP, and 5’-cap can effectively reprogram a number of different human as well as mouse cells. The efficiency even exceeds those by using retrovirus or lentivirus by 10 to 100 fold. Furthermore, the RiPSCs created with mRNAs appear to be closer to hESCs as shown by expression profiling.

Very recently, a few miRNAs that have high expression levels in stem cells were shown to be able to reprogram mouse and human somatic cells when expressed together from a lentivirus [Anokye-Danso et al. 2011]. while that work used lentivirus, thus not directly applicable to the current project, Miyoshi et al. later showed that by using synthesized mature miRNA (overlapping but not the same set of miRNAs as used by Anokye-Danso et al.) reprogramming cold be achieved without viral infection. We believe that this is a promising method and would like to pursue it further and to find out whether these mi-iPSCs relate to hESCs as closely as R-iPSCs. Because transfecting synthetic miRNAs “does operate at considerably lower efficiency” in terms of iPSC creation [Miyoshi et al. 2011],alternatiuve protocols may include transfecting the iPS factor mRNAs together with various miRNAs at different doses and frequencies.

New Product of the Week: EF1a-lacZ lentivirus particles, for expressing nuclear lacZ in virtually any human or mouse cells.

This week save 25% on photoconvertible fluorescent protein mClavGR2 cloning plasmids. Email oligo@allelebiotech.com with code FPBLOG0831.

Blog References: Warren, L. et al. “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA” 2010 Cell Stem Cell 7(5): 618-30

Anokye-Danso, F. et al. “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency” 2011 Cell Stem Cell 8(4): 376-88
Miyoshi, N. et al. “Reprogramming of mouse and human cells to pluripotency using mature microRNAs” 2011 Cell Stem Cell 8(6): 633-8
Kim, H. et al. “miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells” 2011 Cell Stem Cell 8(6): 695-706
Takahashi, K. and Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” 2006 Cell 126(4): 663-76
Yu, J. et al. “Induced pluripotent stem cell lines derived from human somatic cells” 2007 Science 318(5858): 1917-20