Nano Secondaries®纳米二抗

纳米级大小精细成像

Nano Secondaries®是一种可以获得更高的分辨率、更低的背景和更清晰的图像的新型二抗。该二抗是与荧光染料结合的纳米抗体/VHHS,可识别一抗的Fab或Fc片段。

  • 空间位阻小,分辨率极高
  • 通过SRM, IF, WB, FC验证
  • 极小的物种交叉反应性
  • 快速染色:一步孵育
  • 批间高度稳定,重复性高​

Our Nano-Secondary reagents are about 15 kDa in size and thus 10 times smaller than conventional secondary antibodies or IgGs, which are ≥ 150 kDa. That small size considerably improves tissue penetration and minimizes the distance between epitope and label, which increases imaging resolution and can make signals less diffuse. Hence, Nano-Secondary reagents are perfectly suited for super-resolution microscopy techniques like STED, and STORM.

HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and alpaca anti-rabbit IgG VHH Alexa Fluor® 568 (1:1,000). Confocal and gated STED images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and alpaca anti-rabbit IgG VHH Alexa Fluor® 568 (1:1,000). Confocal and gated STED images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

epitope label displacement

Left: primary IgG (dark blue) & Fab and Fc specific Nano secondary reagents (green) with two labels each (red). Right: primary antibody + polyclonal secondary IgG (light blue). The epitope is shown in pink. Epitope label displacement is reduced to a minimum with Nano- Secondary reagents.

During product development we select only Nanobodies with the desired specificity. Nanobody clones with cross-reactivity to other, commonly used species’ IgG or serum proteins are excluded. Therefore, our Nano-Secondary reagents have a very low background and do not require any kind of pre-adsorption. Currently, we offer Nano-Secondary reagents against human IgG, rabbit IgG, and mouse IgG subclasses 1, 2a, 2b, and 3, enabling multiplexing of multiple mouse primary antibodies.

The anti-mouse IgG2b Nano-Secondary is subclass-specific and does not cross-react with IgGs from other commonly used species (here rabbit) and with mouse IgG1 and IgG3 subclasses.

Our nano secondary reagents are subclass-specific and do not cross-react with IgGs from other commonly used species or even same species IgG subtypes.

IF analysis of HeLa cells co-stained with mouse IgG2b anti-tubulin beta antibody and mouse IgG1 CD147 antibody followed by Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue).

Immunofluorescence analysis of HeLa cells co-stained with mouse IgG2b anti-tubulin beta antibody and mouse IgG1 CD147 antibody followed by Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue).

ChromoTek’s recombinant Nano-Secondary reagents are characterized by both DNA and protein sequence. Therefore, we generate identical Nano-Secondary reagents in each manufacturing batch with great purity, and virtually no batch-to-batch variation. The production process of our nanobodies is completely animal free. Through site specific conjugation, we can produce nanobodies with a defined degree of labeling. We validate each batch i.a. with UV/VIS spectroscopy, SDS-PAGE, IF, and mass-spectrometry.

IF analysis of HeLa cells co-stained with mouse IgG2b anti-tubulin beta antibody and mouse IgG1 CD147 antibody followed by Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green) and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue).

ESI-TOF intact weight mass spectrometry validation of the degree of labeling of a Nano-Secondary after site-directed conjugation. The main peak corresponds to the Nano-Secondary with two dyes (expected molecular weight of 18278 Da), indicating >95 % complete and successful labeling.