Alexa Fluor 568 conjugated anti-rabbit IgG, co-stained with Diamond DAPI for nucleus, and Phalloidin 488 for actin.

Fluorescent labeled nano secondary antibodies

Invitrogen Alexa Fluor Nano secondary antibodies* are an exciting, novel class, of fluorescently conjugated antibodies, that offer functional and time saving benefits to basic and translational researchers. Nano secondary antibodies are structurally distinct from traditional antibodies as they are single chain, single domain molecules that can be readily used in microscopy applications, including immunofluorescence or high resolution microscopy. Improve your scientific productivity by reducing the workflow burden, preventing cross-reactivity, and pinpoint your primary target in a diverse set of samples and cell types with Invitrogen Alexa Fluor conjugated Nano secondary antibodies.

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What is a Nano Secondary Antibody?

Nano secondary antibodies or nano bodies were discovered in camelid species such as llamas and alpacas. Nano body molecules retain the antigen binding region of the heavy chain domain of a conventional antibody, but do not have the other light variable chain domains (Figure 1). These single domain antibodies have a small molecular weight (15kDa) and have become popular in research and therapeutic settings, as demonstrated by the recent clinical development of Caplacizumab to treat acquired thrombotic thrombocytopenic purpura (aTTP) (1,2). Their unique structures have provided improved functionality in many ways that makes them useful as secondary antibodies. Invitrogen recombinant Nano secondary antibodies have minimal cross-reactivity to other species IgGs and can be co-incubated with primary antibodies, as well as other Nano secondaries to enable considerable time savings while multiplexing. Ultimately, Invitrogen Nano secondary antibodies provide experimental benefits for fluorescence microscopy.

Alexa Fluor nano secondary conjugated antibody benefits:

  • Co-incubate Alexa Fluor conjugated Nano secondaries with your primary antibody to reduce your workflow by 50% (Figure 2)
  • No cross reactivity to other IgGs of rabbit, rat, sheep, goat, guinea pig, human, and macaque serum
  • Validated for super resolution microscopy, including STED and STORM
  • Shown in a wide range of cell types and models
  • Recombinantly produced antibodies provide lot to lot consistency and stability under various harsh chemical and physical conditions
Recombinant Alexa Fluor conjugated nanobodies

Figure 1. Depiction of Alexa Fluor Nano secondary heavy chain antibodies, that are available in 488, 568 and 647 options. A conventional IgG antibody (left) consists of both heavy and light chain regions, with constant (CH, CL) and variable domain (VH, VL). The single domain variable region of the heavy chain makes up the nanobody molecule (VHH). These single domain nanobody molecules can be generated as recombinant antibodies and then conjugated to Alexa Fluor dyes (right), such as 488, 568 and 647.

Recombinant nano secondary antibodies (VHH) are roughly 1/10th the size of a conventional IgG antibody. Due to their high affinity and minimal cross-reactivity, Alexa Fluor Nano secondary antibodies can be co-incubated with primary antibodies in live cellular models. Therefore, multiple incubation and wash steps are eliminated to reduce the standard imaging protocol by 50% (Figure 2). Importantly these can also have spatial advantages when binding difficult to access epitopes in complex protein structures, providing more subcellular resolution. The depiction in Figure 3 demonstrates the spatial benefits that can enable more secondary molecules to bind their target, improving brightness and resolution of the signal for imaging applications. Explore image gallery below to see fluorescent detection in various immunofluorescence applications.


Reduce immunocytochemistry (ICC) workflow by 50% with Alexa Fluor Nano secondary antibodies

Nanobodies reduce the immunocytochemistry workflow by 50%

Figure 2. ICC workflow comparison using nano secondary antibodies versus conventional secondary antibodies. Co-incubation with Alexa Fluor conjugated Alpaca recombinant nano secondaries eliminates washing and the need for separate incubation in an ICC protocol, which ultimately cuts workflow and sample processing time by 50%.

Nanobodies conjugated to fluorescent molecules binding their primary target

Figure 3. Size comparison of a single domain nano secondary binding its target versus a conventional IgG secondary antibody. Fluorescently conjugated nano secondary antibodies (VHH), such as Alexa Fluor 488 Alpaca anti-rabbit Nano secondary antibody (SA510323), binding both Fab and Fc portions of an IgG primary antibody (left). Traditional fluorescently conjugated secondary antibodies binding to the same target (right) demonstrate that fewer secondary antibody molecules can bind due to their size, when compared to the nano secondary antibodies.

Figure 4. Multiplexed immunostaining with Alexa Fluor 488 Alpaca Nano secondary antibodies, DAPI, and Phalloidin. HeLa cells were simultaneously incubated for 2 hrs at room temperature in 0.1% BSA with primary and secondary antibodies and washed 4 times with PBST before imaging. (A) Primary mouse IgG1 anti-Ku80 (MA512933) was labeled with AF488 Alpaca anti-mouse IgG1 Nano secondary antibody (SA510329). (B)Stained with DAPI for nucleus (blue) and mounted in ProLong Diamond, while (C) was stained for F-Actin using Alexa Fluor 594 Phalloidin (A12381). (D) Composite image showing an overlay of all staining.

Figure 5. Multiplexed immunostaining with Alexa Fluor 568 Alpaca Nano secondary antibodies, DAPI, and Phalloidin. HeLa cells were simultaneously incubated for 2 hrs at room temperature in 0.1% BSA with primary and secondary antibodies and washed 4 times with PBST before imaging. (A) Primary rabbit IgG1 anti-Lamin B1 (PA579606) was labeled with AF568 Alpaca anti-mouse IgG1 Nano secondary antibody (SA510331). (B) Stained with DAPI for nucleus (blue) and mounted in ProLong Diamond, while (C) was stained for F-Actin using Alexa Fluor 488 Phalloidin (A12379). (D) Composite image showing an overlay of all staining.

Figure 9. Confocal and gated STED images of immunostained HeLa cells. Cell were stained with rabbit anti-Lamin B1 antibodies and labeled with Alexa Fluor 568 Alpaca Nano Secondary anti-rabbit IgG. Images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser.

Figure 11. Multiple Alexa Fluor Alpaca Nano Secondary Antibodies applied for multiplex fluorescent Western blotting. Multiple targets analyzed simultaneously on the same blot. Multiplex fluorescent western blot of GFP-TOM70, ß-Tubulin, and GFP in HEK293T cell lysate. Western blot membrane was simultaneously incubated with primary antibodies and Nano-Secondaries. Green: rabbit anti-GFP labeled with Alexa Fluor 488 Nano Secondary anti-rabbit IgG antibody. Magenta: mouse anti-ß-Tubulin labeled with Alexa Fluor 647 alpaca anti-mouse IgG2b.


Alexa Fluor conjugated nano secondary antibody product information

Navigate the tabs below to find a recombinant Alexa Fluor alpaca nano secondary antibody that fit your imaging or western blotting needs.

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510328 Alpaca anti-mouse
IgG1 (Fc) 10 ug
490/525 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510329 Alpaca anti-mouse
IgG1 (Fc) 100 ug
490/525 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510334 Alpaca anti-mouse
IgG2b (Fc) 10 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510335 Alpaca anti-mouse
IgG2b (Fc) 100 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510322 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510323 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510330 Alpaca anti-mouse
IgG1 (Fc) 10 ug
578/603 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510331 Alpaca anti-mouse
IgG1 (Fc) 100 ug
578/603 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510336 Alpaca anti-mouse
IgG2b (Fc) 10 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510337 Alpaca anti-mouse
IgG2b (Fc) 100 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510324 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510325 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510332 Alpaca anti-mouse
IgG1 (Fc) 10 ug
650/665 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510333 Alpaca anti-mouse
IgG1 (Fc) 100 ug
650/665 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510338 Alpaca anti-mouse
IgG2b (Fc) 10 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510339 Alpaca anti-mouse
IgG2b (Fc) 100 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510326 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510327 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510328 Alpaca anti-mouse
IgG1 (Fc) 10 ug
490/525 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510329 Alpaca anti-mouse
IgG1 (Fc) 100 ug
490/525 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510334 Alpaca anti-mouse
IgG2b (Fc) 10 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510335 Alpaca anti-mouse
IgG2b (Fc) 100 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510322 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510323 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
490/525 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510330 Alpaca anti-mouse
IgG1 (Fc) 10 ug
578/603 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510331 Alpaca anti-mouse
IgG1 (Fc) 100 ug
578/603 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510336 Alpaca anti-mouse
IgG2b (Fc) 10 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510337 Alpaca anti-mouse
IgG2b (Fc) 100 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510324 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510325 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
578/603 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

Cat. No. Recombinant Nano Secondary Antibody Ex/Em Applications and Dilution
SA510332 Alpaca anti-mouse
IgG1 (Fc) 10 ug
650/665 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510333 Alpaca anti-mouse
IgG1 (Fc) 100 ug
650/665 IF/ICC: 1:500
Super-resolution microscopy: 1:500
Western blot: 1:1,000
SA510338 Alpaca anti-mouse
IgG2b (Fc) 10 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510339 Alpaca anti-mouse
IgG2b (Fc) 100 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510326 Alpaca anti-rabbit
IgG (Fab, Fc) 10 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000
SA510327 Alpaca anti-rabbit
IgG (Fab, Fc) 100 ug
650/665 IF/ICC: 1:1,000
Super-resolution microscopy: 1:1,000
Western blot: 1:1,000

IgG: Immunoglobulin; Fab: Fragment antigen-binding region; Fc: Fragment crystallizable region; IF: immunofluorescence; ICC: immunocytochemistry

References