Nb600 308

Manufactured by Novus Biologicals

Sourced in United States

The NB600-308 is a piece of laboratory equipment manufactured by Novus Biologicals. It is designed to perform a specific core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation on the intended use. As a result, a more detailed description is not available at this time.

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Close spelling variants of this product

Rabbit anti-GFP NB600-308 antibody Anti-GFP (NB600–308)

23 protocols using nb600 308

Immunofluorescence Staining of Male Germlines

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We performed immunofluorescence staining on dissected male germlines from L4 males grown at 20°. Dissection, fixation and immunofluorescence were performed as described (Jaramillo-Lambert et al. 2007 (link)) with the following alterations. Samples were fixed for 5 min at room temperature in 2% paraformaldehyde in egg buffer [118 mM NaCl, 48 mM KCl2, 2 mM CaCl2, 2 mM MgCl2, 5 mM HEPES at pH 7.4]. We used a 0.7% BSA/1x PBS + 0.1% Tween20 solution for blocking. Rabbit anti-GFP antibody (NovusBiologicals NB600-308) was used at a 1:500 dilution and donkey anti-rabbit antibody Alexa Fluor 488 (Invitrogen A21206) was used at 1:500 for the secondary. DNA was stained with DAPI (final concentration of 0.2 ng/uL). Images were collected with a 63× Plan Apo 1.40 NA objective on an DM6000 epifluorescence compound microscope (Leica) with AF6000 software (Leica). Images were uniformly enhanced using the levels command in Adobe Photoshop.

Herrera L.A, & Starr D.A. (2018). The E3 Ubiquitin Ligase MIB-1 Is Necessary To Form the Nuclear Halo in Caenorhabditis elegans Sperm. G3: Genes|Genomes|Genetics, 8(7), 2465-2470.

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Immunostaining of Embryonic Proteins

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Intact embryos were fixed in 2.5%PFA/ethanol [95 (link)] or methanol/acetone [96 (link)] for all immunofluorescence except for those probed with monoclonal antibody H5, which was fixed in methanol/formaldehyde [95 (link)]. Primary antibodies used were: anti-H3K4me2 (CMA30) 1:1000 END Millipore], anti-P-granules [OIC1D4, 1:5 [96 (link), 97 (link)])], anti-H3K36me3 [(CMA333), 1:1000 [95 (link)]], anti-Ser2p RNA pol II CTD (H5, 1:500, Covance MMS-129R), anti-GFP (1:1000, Novus NB600-308), anti-AMA-1 (1:10,000, Novus 38520002), and anti-FLAG (M2, 1:1000, Sigma F1804). Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-mouse (1:500, Invitrogen R37114) and Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Invitrogen R37117). Samples were mounted in ProLong Gold anti-fade reagent (Life technologies, P36934) and observed under a fluorescence microscope (Leica DMRXA; Hamamatsu Photonics, Hamamatsu, Japan) with Simple PCI software (Hamamatsu Photonics). Image J was used for quantification of raw immunofluorescence intensity [98 ].

Ahn J.H., Rechsteiner A., Strome S, & Kelly W.G. (2016). A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans. PLoS Genetics, 12(8), e1006227.

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Comprehensive Immunochemical Profiling Protocol

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anti-GFP (chicken polyclonal IgY, GFP-1010, Aves Labs, Tigard, OR, USA; 1:2000 for ICC), anti-GFP (rabbit polyclonal, GTX113617, GeneTex, Irvine, CA, USA; 1:2000 for WB), anti-GFP (rabbit polyclonal, NB600-308, Novus Biologicals, Littleton, CO, USA; 1:500 for IP), anti-DDDDK (Flag) (mouse monoclonal, FLA-1, MBL Life Sciences, Tokyo, Japan; 1:10000 for ICC and WB), anti-β-actin (mouse monoclonal, 2D4H5, Proteintech, Rosemont, IL, USA; 1:10000 for WB), anti-α-tubulin (rabbit polyclonal, #11224-1-AP, Proteintech; 1:5000 for WB), anti-TDP-43 (rabbit polyclonal, RN107PW, MBL; 1:1000 for WB), anti-FMP1 (rabbit polyclonal, RN016P, MBL; 1:1000 for WB), anti-PSD95 (mouse monoclonal, 6G6-1C9, Abcam, Cambridge, UK; 1:500 for ICC, 1:3000 for WB), anti-pCofilin (Ser3) (rabbit monoclonal, 77G2, Cell Signaling Technology, Danvers, MA, USA; 1:1000 for WB), anti-pLIMK1 (Thr508)/LIMK2 (Thr505) (rabbit polyclonal, #3841, Cell Signaling Technology; 1:1000 for WB), anti-MAP2(chicken polyclonal IgY, Poly28225, Biolegend, San Diego, CA, USA; 1:10000 for ICC), anti-SMI312 (mouse monoclonal, 837904, Biolegend; 1:1000 for ICC), and anti-Homer1 (rabbit polyclonal, GTX103278, GeneTex; 1:500 for ICC).

Yamada S., Mizukoshi T., Tokunaga A, & Sakakibara S.I. (2022). Inka2, a novel Pak4 inhibitor, regulates actin dynamics in neuronal development. PLOS Genetics, 18(10), e1010438.

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Confocal Imaging of GFP-Expressing Dendritic Cells

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The DCs were treated with 50 μg/ml PNV-GFP for 24 hours in a tissue culture incubator at 5% CO₂, 37 °C, 100% humidity. They were then washed and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Next, the DCs were permeabilized with 0.5% saponin for 15 minutes, then stained for anti-GFP (Novus Biologicals, NB600-308) overnight at 4 °C. Afterwards, the cells were washed and stained with goat anti-rabbit Alexa Fluor 488 conjugated antibody (Jackson ImmunoResearch Laboratories, 111-546-144) for 1 hour at room temperature, followed by nuclear staining with Hoechst dye for 10 minutes. Washed slides were then mounted and imaged with a Zeiss confocal microscope (Digital Microscopy Core, UAMS).

Vang K.B., Safina I., Darrigues E., Nedosekin D., Nima Z.A., Majeed W., Watanabe F., Kannarpady G., Kore R.A., Casciano D., Zharov V.P., Griffin R.J., Dings R.P, & Biris A.S. (2017). Modifying Dendritic Cell Activation with Plasmonic Nano Vectors. Scientific Reports, 7, 5513.

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Multicolor Immunostaining of Lymphoid Tissues

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Lymph nodes were fixed with 4% PFA for two hours, incubated in 30% sucrose, and frozen in O.C.T. Lungs were inflated with, then incubated in 4% PFA for 3 hours, and incubated in 30% sucrose prior to freezing. Eight micron sections were cut and epitopes on slides were stained using tyramide amplification as described ^{21 (link)}. eGFP and YFP were detected using a purified polyclonal rabbit anti-mouse GFP (NB600-308; Novus Biologicals) followed by biotinylated F(ab')₂ donkey anti-rabbit (Jackson ImmunoResearch Laboratories). Biotinylated anti-mouse CD4 (RM4-5; Biolegend); IgD (11-26c; eBiosciences) and FDCM2 (212-MK-2FDCM2; ImmunoKontact) were used to detect CD4 T cells, B cells, and FDC, respectively. 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI; 0.5μg/ml; Roche) in PBS was used to counterstain nuclei prior to mounting on coverslips with Vectashield (Vector laboratories). Images were collected with a Zeiss Axio Imager. Analysis of images was performed using FIJI (ImageJ) software.

Dell'Aringa M, & Reinhardt R.L. (2018). Notch signaling represents an important checkpoint between follicular T-helper and canonical T-helper 2 cell fate. Mucosal immunology, 11(4), 1079-1091.

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Immunoblotting of GFP-tagged proteins

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The youngest fully expanded leaf tissues were weighed, frozen in liquid N₂, and ground in 3 vols of extraction buffer (50 mM HEPES, 100 mM NaCl, 10 mM KCl, and 0.4 M sucrose) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor. Total proteins were denatured by mixing with Laemmli sample buffer and then separated by SDS–PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and probed as described previously (Wang et al., 2011 (link)). Antibodies raised against green fluorescent protein (GFP; NB600-308) were obtained from Novus Biologicals (Littleton, CO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Sade N., Umnajkitikorn K., Rubio Wilhelmi M.D., Wright M., Wang S, & Blumwald E. (2017). Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice. Journal of Experimental Botany, 69(4), 867-878.

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Antibody Panel for Cell Biology

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The following antibodies were used for IB, IF, and IP experiments: anti-Drg1 antibody (dilution 1:1,000 for IB, PAB20044; Abnova), anti-Drg2 antibody (1:1,000 for IB, NBP2-16227; Novus Biological), anti-acetylated tubulin antibody (1:1,000 for IF, T7451; Sigma-Aldrich), monoclonal anti–α-tubulin antibody (1:500 for IF, T9026; Sigma-Aldrich), anti-Dvl2 antibody (1:1000 for IB, 1:200 for IP, 3216S; Cell Signaling Technology), anti-Dvl3 antibody (1:2,000 for IB, 2 µg for IP, 13444–1-AP; Proteintech), anti-phospho-cofilin (Ser3) polyclonal antibody (1:500 for IF, 44-1072G; Thermo Fisher Scientific), anti-phospho LIMK1 (phospho T508) antibody (1:500 for IF, ab38508; Abcam), anti-GAPDH antibody (1:5,000 for IB, FL-335; Santa Cruz Biotechnology), anti-GFP antibody (1 µl/tube for IP, 1:500 for IF, NB600-308; Novus Biological), anti-GST antibody (1:2,000 for IB, ab19256; Abcam), HA-tag (C29F4) antibody (1:1,000 for IF, 3724; Cell Signaling Technology), anti-V5-peroxidase antibody (dilution 1:10,000 for IB, V2260; Sigma-Aldrich), and monoclonal anti-Flag-M2 HRP antibody (dilution 1:5,000 for IB, A8592; Sigma-Aldrich).

Lee M., Hwang Y.S., Yoon J., Sun J., Harned A., Nagashima K, & Daar I.O. (2019). Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled. The Journal of Cell Biology, 218(8), 2659-2676.

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Immunohistochemical Enhancement of Anterograde Tracing

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To amplify synaptophysin densities in the anterograde tracing experiments, the GFP signal was enhanced by antibody staining. Perfused tissue was sliced on a freezing microtome at 60 µM, and every third section throughout the brain was stained. Briefly, sections were rinsed in PBS with 0.1% Triton (PBST), then blocked in 1.5% normal horse serum diluted in PBST for 1 hr (blocking solution) at room temperature. Primary antibody Rrabbit anti-GFP (Novus Biologicals NB600-308) was diluted 1:2000 in blocking solution and sections were incubated overnight at 4°C. After rinsing with PBST, secondary anti-rabbit-A488 was applied at 1:200 dilution in blocking buffer and incubated overnight at 4°C. Sections were rinsed in PBS, mounted to superfrost plus slides, coverslipped with Vectashield containing DAPI, and imaged (see details above).

Autry A.E., Wu Z., Kapoor V., Kohl J., Bambah-Mukku D., Rubinstein N.D., Marin-Rodriguez B., Carta I., Sedwick V., Tang M, & Dulac C. (2021). Urocortin-3 neurons in the mouse perifornical area promote infant-directed neglect and aggression. eLife, 10, e64680.

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Multicolor Immunostaining of Lymphoid Tissues

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Dell'Aringa M, & Reinhardt R.L. (2018). Notch signaling represents an important checkpoint between follicular T-helper and canonical T-helper 2 cell fate. Mucosal immunology, 11(4), 1079-1091.

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Protein-Nucleosome Interaction Assay

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MyOne Streptavidin T1 magnetic beads (Invitrogen) were aliquoted (20 μl/tube) and washed with binding / wash buffer (20 mM Tris HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 5% glycerol and 1× protease inhibitors). To pre-conjugate beads with recombinant nucleosomes (Epicypher), 2 μg of biotinylated nucleosomes was incubated with the equilibrated streptavidin beads in 600 μl binding buffer for 1 hours at 4 °C with constant rocking. After incubation, the beads were washed with binding / wash buffer. 3 μg GFP fusion proteins were then added and incubated with peptide-beads in 600 μl binding buffer for 3 hours at 4 °C. After three washes with wash buffer, the beads were resuspended in 40 μl 2× SDS loading buffer and boiled for 6–8 min. 10 μl of pull-down sample was analyzed by western blot with an anti-GFP antibody (1:2,000, Novus, NB600-308).

Gu T., Hao D., Woo J., Huang T.W., Guo L., Lin X., Guzman A.G., Tovy A., Rosas C., Jeong M., Zhou Y., Deneen B., Huang Y., Li W, & Goodell M.A. (2022). The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development. Nature genetics, 54(5), 625-636.

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