Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.
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References
1.
PoppMW, AntosJM, GrotenbregGM, SpoonerE, and PloeghHL: Sortagging: a versatile method for protein labeling. Nat Chem Biol, 2007; 3:707–708.
2.
BallardJL, PeevaVK, deSilvaCJS, LynchJ, and SwansonNR: Comparison of Alexa Fluor and CyDye for practical DNA microarray use. Mol Biotechnol, 2007; 36:175–183.
3.
Alexa Fluor dyes spanning the visible and infrared spectrum—Section 1.3. Life Technologies. Available at: http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/fluorophores-and-their-amine-reactive-derivatives/alexa-fluor-dyes-spanning-the-visible-and-infrared-spectrum.html
4.
BerlierJE, RotheAA, BullerGG, BradfordJJ, GrayDR, FilanoskiBJ, TelfordWG, YueSS, LiuJJ, CheungCY, ChangWW, HirschJD, BeechemJM, HauglandRP, and HauglandRP: Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem, 2003; 51:1699–1712.
5.
LoveJC, RonanJL, GrotenbregGM, van der VeenAG, and PloeghHL: A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol, 2006; 24:703–707.
6.
NimanHL, HoughtenRA, WalkerLE, ReisfeldRA, WilsonIA, HogleJM, and LernerRA: Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci USA, 1983; 80:4949–4953.
7.
MendozaVL, AntwiK, Barón-RodríguezMA, BlancoC, and VachetRW: Structure of the preamyloid dimer of beta-2-microglobulin from covalent labeling and mass spectrometry. Biochemistry, 2010; 49:1522–1532.
8.
YamanakaYJ, SzetoGL, GierahnTM, ForcierTL, BenedictKF, BrefoMS, LauffenburgerDA, IrvineDJ, and LoveJC: Cellular barcodes for efficiently profiling single-cell secretory responses by microengraving. Anal Chem, 2012; 84:10531–10536.
9.
VaradarajanN, KwonDS, LawKM, OgunniyiAO, AnahtarMN, RichterJM, WalkerBD, and LoveJC: Rapid, efficient functional characterization and recovery of HIV-specific human CD8+T cells using microengraving. Proc Natl Acad Sci USA, 2012; 109:3885–3890.
10.
CastilloFJ, MullenLJ, GrantBC, DeLeonJ, ThriftJC, ChangLW, IrvingJM, and BurkeDJ: Hybridoma stability. Dev Biol Stand, 1994; 83:55–64.
11.
KrishnanIS, HansenHJ, GoldDV, GoldenbergDM, and LeungSO: Co-secretion of two distinct kappa light chains by the mu-9 hybridoma. Hybridoma, 1999; 18:325–333.
12.
Payelle-BrogardB, RagimbeauJ, AvrameasS, and ChristodoulouC: Immunoglobulin double isotype-producing hybridomas isolated from an autoimmune (NZB x NZW) F1 mouse. Hybridoma, 1998; 17:289–297.
13.
ZackDJ, WongAL, StempniakM, and WeisbartRH: Two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma: implications for isotypic exclusion. Mol Immunol, 1995; 32:1345–1353.
14.
ParekhR, IsenbergD, RookG, RoittI, DwekR, and RademacherT: A comparative analysis of disease-associated changes in the galactosylation of serum IgG. J Autoimmun, 1989; 2:101–114.
15.
WeiAP, and HerronJN: Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding. J Mol Recog, 2002; 15:311–320.
16.
Anti dye and anti hapten antibodies—Section 7.4. Life Technologies. Available at: http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/antibodies-avidins-lectins-and-related-products/anti-dye-and-anti-hapten-antibodies.html#head2
17.
tools.lifetechnologies.com. Available at: http://tools.lifetechnologies.com/content/sfs/manuals/mp06398.pdf
18.
van RijnsoeverC, OorschotV, and KlumpermanJ: Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods, 2008; 5:973–980.
19.
KöhlerG, and MilsteinC: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975; 256:495–497.
20.
PoppMW, AntosJM, and PloeghHL: Site-specific protein labeling via sortase-mediated transpeptidation. Curr Protocol Protein Sci Ch 15, Unit 15:3, 2009.
21.
GuimaraesCP, WitteMD, TheileCS, BozkurtG, KundratL, BlomAEM, and PloeghHL: Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat Protocol, 2013; 8:1787–1799.