...of basal H 2 O 2 levels with peroxiredoxin-based probes Real-time monitoring of basal H 2 O 2 levels with...Belousov Hydrogen Peroxide (H 2 O 2 ) Cytosolic or mitochondrial sensor for H 2 O 2 oxidation (roGFP2-Orp1) ...peroxiredoxin-2-based probe. Nat Commun. 2018 Aug 7;9(1):3145. Hadley Sikes Hydrogen Peroxide (H 2 O 2 ) Monitoring...Calcium D and D2 family Cameleon calcium sensors Bcl-2-mediated alterations in endoplasmic reticulum Ca2...Calcium Red fluorescent calcium sensors (fRCaMP1/2, GECO1/2) The kinetic mechanisms of fast-decay red-fluorescent...intracellular Zn(2+) homeostasis. Nat Methods. 2009 Oct;6(10):737-40. Maarten Merkx Zinc eZinCh-2 Zn2+ FRET ... eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging...

...CCNB1 CCNB2 CCNB3 Cyclin B1, 2, or 3 Cyclin D CCND1 CCND2 CCND3 Cyclin D1, 2, or 3 Cyclin E CCNE1 CCNE2 ...kinase 1 CHK2 Checkpoint kinase 2 Cop-1 Ring finger and WD repeat domain 2 (RFWD2); E3 ubiquitin protein...SESN3 Sestrins 1, 2, or 3 Siah Siah E3 ubiquitin protein ligase 1 TSC2 Tuberous sclerosis 2 TSP1 Thrombospondin... Spring Harbor Perspectives in Biology. 2010 Feb;2(2):a001107. PMC PMID: 20182618 . Do you have suggestions...threonine kinase ATR ATR serine/threonine kinase B99 G-2 and S-phase expressed 1; also known as GTSE1 BAI-1...inhibitor 1A p48 Damage-specific DNA binding protein 2, 48kDa p53 Tumor protein p53 p53AIP1 Tumor protein... domain protein 1 PIGs Etoposide induced 2.4 PIRH-2 Ring finger and CHY zinc finger domain containing ...

...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...Rabbits have only one IgG subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...

...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...transcribed into cDNA fragments that are efficiently subcloned into the phagemid pDX_init plasmid, using the ...sybody pools from the phage display screens are subcloned (using FX cloning) into the expression vector ...for identification. All unique sybodies can be subcloned into pBXNPH3 or pBXNPHM3 expression plasmids for...

...FHF2 Human Mouse IgG2a 114527 Anti-JIP-2/IB-2 [N135/37.2R] JIP-2/IB-2 Mouse Mouse IgG2a 114529 Anti-Pan-SAPAP...Anti-CDY1/2/L1/L2 [N143/36R] CDY1/2/L1/L2 Human Mouse IgG2a 206535 Anti-CDY1/2 [N143/38R] CDY1/2 Human Mouse...206763 Neuroligin-2 scFv [L107/90] L107/90 scFv Neuroligin-2 Mouse Mouse 206764 Neuroligin-2 scFv [L107/95...receptor Rat Mouse IgG2a 114484 Anti-Stonin-2 [N346/9R] Stonin-2 Human Mouse IgG2a 114485 Anti-NSD3 [N348...Pan-FHF-A Human Mouse IgG2a 114510 Anti-REEP1/2 [N326D/29R] REEP1/2 Mouse Mouse IgG2a 114511 Anti-Kv3.1b K+ ...Mouse Mouse IgG2a 114547 Anti-Mitofusin-2 [N171/17.4R] Mitofusin-2 Mouse Mouse IgG2a 114548 Anti-Mortalin...Mouse IgG2a 140065 Anti-SCG10/Stathmin-2 [L5/1R] SCG10/Stathmin-2 Rat Mouse IgG2a 140066 Anti-Co-Rest/RCOR1...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody...antibodies contain a mix of different isotypes and subclasses. They each have their own advantages. Since monoclonal...species, like an anti-mouse IgG antibody. Some are subclass specific, like an anti-mouse IgG2a antibody. When...    Figure 1: Broadly reactive versus subclass-specific antibodies. Only a fraction of the antibody... antibody (A). To boost signal, consider using subclass specific secondary antibodies instead (B). ...antibody contains a mix of different isotypes and subclasses, use a broadly reactive secondary antibody to...antibody you may wonder, “Will the isotype, and subclass affect my experiment?” Well, it depends on the...

...immunoglobulin heavy diversity 2-15 D2, IGHD215 IGHD2-2 immunoglobulin heavy diversity 2-2 IGHD22 IGHD2-21 immunoglobulin...heat shock 70kDa protein 2 HSP70-2, HSP70-3 HSPA4 heat shock 70kDa protein 4 APG-2, HS24/P52, MGC131852, ...variable 2-33 (non-functional) IGLV233, V1-9 IGLV2-8 immunoglobulin lambda variable 2-8 IGLV28, V1-2 IGLV3...beta 4 DEFB-2, DEFB102, DEFB2, HBD-2, SAP1 EDN1 endothelin 1 ET1, HDLCQ7 EDN2 endothelin 2 ET2, PPET2 ...receptor subfamily 2, group C, member 1 TR2 NR2C2 nuclear receptor subfamily 2, group C, member 2 TAK1, TR2R1...suppression of tumorigenicity 2 - STC1 stanniocalcin 1 STC STC2 stanniocalcin 2 STC-2, STCRP TAC1 tachykinin,...HPS, HPS2, PE AZGP1 alpha-2-glycoprotein 1, zinc-binding ZA2G, ZAG B2M beta-2-microglobulin - CALR calreticulin...

...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch... be included in a single PCR, for example for subcloning genes from different templates. All PCRs are ...can be included at the 5’ end of either primer. Subcloning - To move a gene from one vector to another simply...

...multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant antibodies...different subclasses, but there are also secondary antibodies that are specific to each subclass. Approximately...mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass specific secondary...backbone to generate R-mAbs of different subclasses, including subclasses that were not produced by the original...original hybridoma. By using subclass specific secondary antibodies and subclassed switched R-mAbs, multiple...engineered. Like humans, mice express multiple subclasses of IgG antibody. Broad range secondary antibodies...KD, Engebrecht J, Trimmer JS. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...antibodies can be isotype-specific or even isotype subclass-specific. This specificity can be used to ensure...Isotype Description Configuration and Valency Subclasses IgA Prevalent antibody in secretions (e.g. tears...

...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh...Protocols, 2016 . Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh...PX458_ARID4B_1 BCL6_iso1 Human FLAG pFETCh_BCL6_iso1 PX458_BCL6_iso1_1 PX458_BCL6_iso1_2 CEBPG Human FLAG...

...mouse somatic cells. Cell Stem Cell. 2008 Feb 7. 2(2):151-9. Jaenisch Lentivirus Mouse Polycistronic, ...Nature of Induced Pluripotency. Cell. 2015 Jul 16;162(2):412-24. Mikkelsen Lentivirus Human Expression of ...self-replicative RNA. Cell Stem Cell. 2013 Aug 1;13(2):246-54. Dowdy Adenovirus Mouse Non-integrating expression...reprogramming in mouse. Cell Stem Cell. 2008 Mar 6. 2(3):230-40. Hochedlinger MMLV-derived Retrovirus Mouse...Lentiviral Human Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic...Conversion of Fibroblasts. Neuron. 2014 Oct 22;84(2):311-23. Yoo Fibroblasts Neurons Lentiviral Human ...

...otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult... from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can be ...be from the same host species, have the same Ig subclass, and be in the same fluorophore (e.g., if you ...staining for anti-mouse Ly6C in APC with the IgG2c subclass, you will use the APC IgG2c isotype control). ...

...IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the...and α. Some isotypes have different versions or subclasses that have minor differences in their heavy chain...the hinge region in IgM antibodies. IgM has no subclasses and is found mostly in blood. IgD While IgD is...monomer structure with a valency of two and has no subclasses. IgG IgG is the most abundant isotype in blood...monomer with a valency of two. There are four IgG subclasses, numbered based on their abundance in blood: ...involved in allergic reactions. There are no IgE subclasses and they are expressed as monomers with a valency...dimer with a valency of four. There are two IgA subclasses: IgA1 and IgA2.  You can learn more about isotype...

...to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID ...cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control Rho GTPase activity. A) Schematic...Rho GTPases based on BcLOV4. These single-component systems take advantage of BcLOV4’s light-induced binding...light iLID-SspB heterodimerizes under blue light BcLOV4 undergoes a conformational change and binds the...

... been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (like...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV ... own subclone, they were able to develop a 12-plex multiplex imaging system. Mixing the subclones within...their work with the “signalome,” a 12-plex mixed-subclone system using mStrawberry, localization peptides...

...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y,...Y, Lin C, et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin...

...MO, Trono D, Aebischer P. Nat Methods. 2006. Feb;3(2):109-16. PubMed . In vivo RNA interference demonstrates...(CAG promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) On Patrick Aebischer and Didier Trono...(CAG promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) Off Patrick Aebischer and Didier ...1alpha promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) On Patrick Aebischer and Didier Trono...hPGK promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) On Patrick Aebischer and Didier Trono...hPGK promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) Off Patrick Aebischer and Didier ...hUbiquitin promoter) ‐ OR ‐ shRNA (H1 promoter when subcloned from pLVTHM) On Patrick Aebischer and Didier Trono...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...