Nano-Secondary® reagents
Nano-Secondary® reagents are highly specific recombinant alpaca single domain VHHs (also known as Nanobodies) that recognize Fab or Fc fragments of primary antibodies. Their small size leads to minimal epitope-label displacement, resulting in sharper images and higher resolution, making them highly suitable for super resolution imaging applications (SRM).
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Higher resolution due to minimal label displacement
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Excellent reproducibility due to high lot-to-lot consistency
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Minimal cross-reactivity
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Validated for SRM, IF, WB , FC
Available Nano-secondary reagents
![HeLa cells were immunostained with anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (grey). Nuclei were stained with DAPI, blue. Scale bar, 10 μm.](https://www.ptglab.com/products/pictures/sms1AF647-1-7.jpg)
HeLa cells were immunostained with anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (grey). Nuclei were stained with DAPI, blue. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Multiplexed immunostaining of HeLa cells with two alpaca anti-mouse Nano-Secondaries and one anti-rabbit Nano-Secondary. Green: mouse IgG1 anti-COX4 + alpaca anti-mouse IgG1 VHH Alexa Fluor 488. Magenta: mouse IgG2b anti-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor 647. Yellow: rabbit anti-Lamin + alpaca anti-rabbit IgG VHH Alexa Fluor 568. Scale bar, 10 μm.](https://www.ptglab.com/products/pictures/sms1AF488-1-5.jpg)
Multiplexed immunostaining of HeLa cells with two alpaca anti-mouse Nano-Secondaries and one anti-rabbit Nano-Secondary. Green: mouse IgG1 anti-COX4 + alpaca anti-mouse IgG1 VHH Alexa Fluor 488. Magenta: mouse IgG2b anti-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor 647. Yellow: rabbit anti-Lamin + alpaca anti-rabbit IgG VHH Alexa Fluor 568. Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-vimentin antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green). Nuclei were stained with DAPI (blue).](https://www.ptglab.com/products/pictures/Result-smsG1CL488-1.jpg)
Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-vimentin antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-CD147 antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.](https://www.ptglab.com/products/pictures/Result-smsG1CL647-1.jpg)
Immunofluorescence analysis of HeLa cells stained with mouse IgG1 anti-CD147 antibody and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of HeLa cells stained with mouse IgG2a anti-Actin antibody and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488 (green) Nuclei were stained with DAPI (blue).](https://www.ptglab.com/products/pictures/Result-smsG2aCL488-1.jpg)
Immunofluorescence analysis of HeLa cells stained with mouse IgG2a anti-Actin antibody and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488 (green) Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of Hela cells stained with mouse IgG2a anti-Tubulin antibody (66240-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (magenta). Nuclei were stained with DAPI (blue).](https://www.ptglab.com/products/pictures/Result-smsG2aCL647-1.jpg)
Immunofluorescence analysis of Hela cells stained with mouse IgG2a anti-Tubulin antibody (66240-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (magenta). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of HeLa cells stained with mouse IgG2b anti-Tubulin beta antibody and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, orange). Nuclei were stained with DAPI (blue).](https://www.ptglab.com/products/pictures/Result-smsG2bCL555-1.jpg)
Immunofluorescence analysis of HeLa cells stained with mouse IgG2b anti-Tubulin beta antibody and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, orange). Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of HeLa cells co-stained with mouse IgG1 anti-HSP60 antibody (66041-1-Ig) and mouse IgG2b anti-Lamin A/C antibody followed by Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, yellow) and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta).](https://www.ptglab.com/products/pictures/Result-smsG1CL555-1_20230720151733936.jpg)
Immunofluorescence analysis of HeLa cells co-stained with mouse IgG1 anti-HSP60 antibody (66041-1-Ig) and mouse IgG2b anti-Lamin A/C antibody followed by Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, yellow) and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, magenta). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of A431 cells stained with rabbit anti-COXIV antibody (11242-1-AP, magenta) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 647 (srb2GCL647-1), 1:500. Nuclei were stained with DAPI (blue).](https://www.ptglab.com/products/pictures/Result-srb2GCL647-1_20240226162005292.gif)
Immunofluorescence analysis of A431 cells stained with rabbit anti-COXIV antibody (11242-1-AP, magenta) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 647 (srb2GCL647-1), 1:500. Nuclei were stained with DAPI (blue). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Immunofluorescence analysis of A431 cells stained with rabbit anti-tubulin antibody (80713-1-AP, cyan) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 750 (srb2GCL750-1), 1:500. Nuclei were stained with DAPI.](https://www.ptglab.com/products/pictures/Result-srb2GCL750-1_20240226162553961.gif)
Immunofluorescence analysis of A431 cells stained with rabbit anti-tubulin antibody (80713-1-AP, cyan) and Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, CoraLite® Plus 750 (srb2GCL750-1), 1:500. Nuclei were stained with DAPI. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
![Live A431 cells were incubated with anti-human EGFR (Cetuximab biosimilar) followed by Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 555 [CTK0117] (shuGCL555-2, orange). Cells were fixed and nuclei were stained with DAPI (blue). Epifluorescence images were acquired with a 40x objective and post-processed.](https://www.ptglab.com/products/pictures/Result-shuGCL555-2.jpg)
Live A431 cells were incubated with anti-human EGFR (Cetuximab biosimilar) followed by Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 555 [CTK0117] (shuGCL555-2, orange). Cells were fixed and nuclei were stained with DAPI (blue). Epifluorescence images were acquired with a 40x objective and post-processed.
![Immunofluorescence analysis of transgenic CHO-TIM3 cells. PFA-fixed cells were stained with anti-human TIM3 (Cobolimab biosimilar), Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 647 [CTK0117] (gray) and DAPI (blue).](https://www.ptglab.com/products/pictures/Result-shuGCL647-2_20231123151955368.jpg)
Immunofluorescence analysis of transgenic CHO-TIM3 cells. PFA-fixed cells were stained with anti-human TIM3 (Cobolimab biosimilar), Nano-Secondary® alpaca anti-human IgG, recombinant VHH, CoraLite® Plus 647 [CTK0117] (gray) and DAPI (blue).
![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.](https://www.ptglab.com/products/pictures/srbAF568-1-4.jpg)
![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.](https://www.ptglab.com/products/pictures/srbAF568-1-4.jpg)
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.
![Vimentin in HeLa cells: The cells were stained with monoclonal anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 488 and recorded with Leica GSDIM system.](https://www.ptglab.com/products/pictures/sms1AF488-1-3.jpg)
Vimentin in HeLa cells: The cells were stained with monoclonal anti-Vimentin mouse IgG1 antibody and anti-mouse IgG1 VHH Alexa Fluor® 488 and recorded with Leica GSDIM system. The image is a courtesy of Dr. Leila Nahidiazar, Dr. Jop Kind, and Prof. Kees Jalink.
![Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with anti-GFP mouse IgG 1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647.](https://www.ptglab.com/products/pictures/sms1AF647-1-3.jpg)
![Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with anti-GFP mouse IgG 1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647.](https://www.ptglab.com/products/pictures/sms1AF647-1-3.jpg)
Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with anti-GFP mouse IgG 1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647.
![Western blot analysis of endogenous ®-Tubulin in HEK293T cell lysate. Detection with mouse anti-®-Tubulin antibody and alpaca anti-mouse IgG2b VHH Alexa Fluor® 647.](https://www.ptglab.com/products/pictures/sms2bAF647-1-8.jpg)
Western blot analysis of endogenous ®-Tubulin in HEK293T cell lysate. Detection with mouse anti-®-Tubulin antibody and alpaca anti-mouse IgG2b VHH Alexa Fluor® 647.
![HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-Lamin B1 (66095-1-Ig), anti-HNRNPA (67445-1-Ig), and anti-Tom20 (66777-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, blue), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 750 (smsG2aCL750-1, white), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green).](https://www.ptglab.com/products/pictures/Result-smsG1CL647-1_20230720162018716.jpg)
HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-Lamin B1 (66095-1-Ig), anti-HNRNPA (67445-1-Ig), and anti-Tom20 (66777-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 647 (smsG1CL647-1, blue), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 750 (smsG2aCL750-1, white), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 488 (smsG2bCL488-1, green).
![HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-ATP50 (66696-1-Ig), anti-AIF (67791-1-Ig), and anti-actin (66009-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 750 (smsG1CL750-1, white), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488 (smsG2aCL488-1, green), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, red).](https://www.ptglab.com/products/pictures/Result-smsG2bCL555-1_20230721090049336.jpg)
HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-ATP50 (66696-1-Ig), anti-AIF (67791-1-Ig), and anti-actin (66009-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 750 (smsG1CL750-1, white), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488 (smsG2aCL488-1, green), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 555 (smsG2bCL555-1, red).
![HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-TDP43 (60019-1-Ig), anti-tubulin (66240-1-Ig), and anti-IMP3 (66247-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, red), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (smsG2aCL647-1, blue), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 750 (smsG2bCL750-1, white).](https://www.ptglab.com/products/pictures/Result-smsG1CL555-1_20230720151807625.jpg)
HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-TDP43 (60019-1-Ig), anti-tubulin (66240-1-Ig), and anti-IMP3 (66247-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 555 (smsG1CL555-1, red), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647 (smsG2aCL647-1, blue), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 750 (smsG2bCL750-1, white).
![HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-PCNA (60097-1-Ig), anti-cytochrome C (66264-1-Ig), and anti-alpha tubulin (66031-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 555 (smsG2aCL555-1, red), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG2bCL647-1, blue).](https://www.ptglab.com/products/pictures/Result-smsG2bCL647-1_20230721114628502.jpg)
HEK-293 cell lysates were subjected to SDS-PAGE followed by multiplex western blot analysis with 3 mouse primary antibodies including anti-PCNA (60097-1-Ig), anti-cytochrome C (66264-1-Ig), and anti-alpha tubulin (66031-1-Ig). Primary antibodies were detected using 3 mouse IgG subclass-specific nano-secondary reagents including Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, CoraLite® Plus 488 (smsG1CL488-1, green), Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 555 (smsG2aCL555-1, red), and Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, CoraLite® Plus 647 (smsG2bCL647-1, blue).
![Immunostaining with Alpaca anti-mouse IgG1 Alexa Fluor 647 Nano-Secondaries for FACS. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 + alpaca anti-mouse IgG1 VHH Alexa Fluor 647 (1:600). CD3- cells were pre-stained with Calcein. Two cell populations can be clearly distinguished on the dot-plot: Alexa Fluor 647-positive Calcein-negative cells and Alexa Fluor 647-negative Calcein-positive cells.](https://www.ptglab.com/products/pictures/sms1AF647-1-10.jpg)
Immunostaining with Alpaca anti-mouse IgG1 Alexa Fluor 647 Nano-Secondaries for FACS. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 + alpaca anti-mouse IgG1 VHH Alexa Fluor 647 (1:600). CD3- cells were pre-stained with Calcein. Two cell populations can be clearly distinguished on the dot-plot: Alexa Fluor 647-positive Calcein-negative cells and Alexa Fluor 647-negative Calcein-positive cells.
![Flow cytometry with anti-mouse Nano-Secondaries. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (1:600). Two cell populations can be clearly distinguished with a 2-log shift: CD3-positive cells (in green) and CD3-negative cells (in grey).](https://www.ptglab.com/products/pictures/sms1AF647-1-9.jpg)
Flow cytometry with anti-mouse Nano-Secondaries. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (1:600). Two cell populations can be clearly distinguished with a 2-log shift: CD3-positive cells (in green) and CD3-negative cells (in grey).
![Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488.](https://www.ptglab.com/products/pictures/Result-smsG2aCL488-1_20230608095914818.jpg)
Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 488.
![Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 555.](https://www.ptglab.com/products/pictures/Result-smsG2aCL555-1_20230608102307314.jpg)
Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 555.
![Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647.](https://www.ptglab.com/products/pictures/Result-smsG2aCL647-1_20230608113340739.jpg)
Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 647.
![Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 750.](https://www.ptglab.com/products/pictures/Result-smsG2aCL750-1_20230608160542333.jpg)
Flow cytometry analysis of 1X10^6 human peripheral blood mononuclear cells (PBMCs) stained with anti-human CD3 (clone OKT3, 65133-1-Ig) and Nano-Secondary® alpaca anti-mouse IgG2a, recombinant VHH, CoraLite® Plus 750.
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.](https://www.ptglab.com/products/pictures/srbAF568-1-4.jpg)
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](https://www.ptglab.com/media/ppsklddk/epitope-label-displacement.png)
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.](https://www.ptglab.com/media/dkflduyw/nano-secondaries-cross-reactivity.png)
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).](https://www.ptglab.com/products/pictures/Result-smsG2bCL488-1_20230721083959674.jpg)
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).](https://www.ptglab.com/media/mbigynxc/esi-tof-intact-weight-mass-spectrometry-validation.png)
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.