Alexa Fluor Conjugated Nanobodies | Thermo Fisher Scientific

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 (C_H, C_L) and variable domain (V_H, V_L). The single domain variable region of the heavy chain makes up the nanobody molecule (V_HH). 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 (V_HH) are roughly 1/10^th 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 (V_HH), 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.

Image gallery of Alexa Fluor conjugated Alpaca recombinant Nanobodies

Fluorescent staining using Alexa Fluor 488 Nano secondary antibodies and imaging reagents.

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.

Fluorescent staining using Alexa Fluor 568 Nano secondary antibodies and imaging reagents

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 6. One-step immunostaining (top row) vs. sequential immunostaining (bottom row) of HeLa cells. Anti-β-Actin (left column) and anti-LaminB1 (right column) primary rabbit antibodies labeled with Alexa Fluor 647 Alpaca anti-rabbit IgG Nano secondary antibody (SA510327). Scale bar, 20 μm.

Multiplexed immunostaining of HeLa cells with Alexa Fluor 647 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1, and Alexa Fluor 568 anti-rabbit IgG

Figure 7. Multiplex imaging of Hela cells with three Alexa Fluor Alpaca Nano Secondary antibodies. Primary mouse IgG1 anti-COX4 labeled with Alexa Fluor 488 Nano anti-mouse IgG1 Nano secondary antibody (green) (SA510329). Primary mouse IgG2b anti-Tubulin labeled with Alexa Fluor 647 anti-mouse IgG2b Nano secondary antibody (purple) (SA510339). Primary rabbit anti-Lamin labeled with Alexa Fluor 568 anti-rabbit IgG Nano secondary antibody (yellow) (SA510325). Scale bar, 10 μm.

Alexa Fluor 647 anti-rabbit IgG nanobody

Figure 8. Immunostaining of HeLa cells. Stably expressing Tubulin-GFP cells were stained with rabbit anti-GFP PABG1 antibody and Alexa Fluor 647 anti-rabbit IgG Nano secondary antibody (SA510327). Scale bar, 10 μm.

Confocal and STED images stained with Alexa Fluor 647 Nano Secondary antibody

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.

Western blot analysis using Alexa Fluor 647 secondary nanobody

Figure 10. Western blot analysis of endogenous ß-Tubulin in HEK293T cell lysate. HEK293T cell lysates probed for ß-Tubulin with primary mouse anti-ß-Tubulin antibody. Alexa Fluor 647 Alpaca Nano Secondary anti-mouse IgG2b antibody was used for labeling.

Multiplexed Fluorescent western blot analysis using Alexa Fluor 647 secondary nanobody

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