UltraTek Anti-Maus
Polyclonal anti-immunoglobulin G (anti-IgG) secondary antibodies are indispensable tools for many molecular biology techniques and diagnostic tests. However, their animal production is a major ethical problem. Here we present a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced on a large scale in Escherichia coli and could make secondary antibody production in animals obsolete. Their recombinant nature allows for fusion with affinity tags or reporter enzymes, as well as efficient maleimide chemistry for fluorophore coupling.
We demonstrate their superior performance in Western blotting in both peroxidase- and fluorophore-bound forms. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and high-resolution microscopy with much less label shift than traditional secondary antibodies. They also allow for simpler and faster immunostaining protocols and enable multi-target localization using primary IgGs of the same species and class.
introduction
Mouse and rabbit antibodies are fundamental tools for many basic research techniques and medical diagnostic assays. These primary antibodies are usually detected or immobilized indirectly via polyclonal anti-IgG secondary antibodies.
- The need for a continuous supply of anti-IgG sera necessitates the maintenance, immunization, exsanguination and ultimately culling of large numbers of goats, sheep, rabbits and donkeys, which is not only costly but also poses a major welfare and ethical issue (Shen, 2013; Reardon, 2016).
- In addition, each new batch of serum contains another heterogeneous mixture of antibodies that have to be affinity-purified on IgG columns and then freed (by pre-adsorption) of non-specific and cross-reacting antibodies. In addition, the success of this process must be quality checked each time.
- The large size of secondary antibodies (∼10–15 nm; 150 kD) is also a disadvantage as it limits tissue penetration and introduces considerable label displacement, reducing the image resolution achievable by super-resolution fluorescence microscopy methods (Ries et al., 2012; Szymborska et al., 2013; Pleiner et al., 2015). Furthermore, their non-recombinant nature precludes genetic engineering (tagging or fusion with reporter enzymes).
- Why then have recombinant anti-IgG detection reagents not replaced secondary polyclonal antibodies? The main problem is the signal strength. For example, the signal in traditional immunofluorescence is enhanced by (a) multiple secondary IgG molecules binding to different epitopes of a primary antibody; (b) a large IgG that tolerates many labels per molecule; and (c) their bivalent binding mode, which exploits avidity for high-affinity target recognition. Given these facts, it seems very challenging to achieve comparable signal levels with a small, monovalent, monoclonal reagent.
We considered nanobodies, single domain antibodies derived from camelid heavy chain antibodies (Hamers-Casterman et al., 1993; Arbabi Ghahroudi et al., 1997; Muyldermans, 2013), as perhaps the best candidates for such reagents.
Because of their small size (∼3 × 4 nm; 13 kD), possibility of their renewable production as recombinant fusion proteins, and favorable biophysical properties, nanobodies have attracted considerable attention as powerful tools in cell biology ( Helma et al., 2015 ) and structural biology (Desmyter et al., 2015) and as future therapeutics ( Van Bockstaele et al., 2009; Kijanka et al., 2015). ). They are particularly useful for high-resolution imaging(Ries et al., 2012; Szymborska et al., 2013; Pleiner et al., 2015; Göttfert et al., 2017; Traenkle and Rothbauer, 2017).
The resolving power of some of the best microscopes reported so far (e.g. ∼6 nm by Balzarotti et al. [2017] ; ∼10–20 nm by Xu et al. [2012] or Huang et al. [2016]) can be seen as a result of the offset between fluorescent label and target introduced by primary and secondary antibodies (20-30 nm). Site-specifically labeled nanobodies represent a promising solution to this problem, as they can place fluorophores closer than 2 nm to their antigen and, despite their small size, even tolerate up to three dyes (Pleiner et al., 2015).
In this study, we describe the generation of a comprehensive toolbox of nanobodies against all mouse IgG subclasses and rabbit IgG. This work required very extensive optimizations to our routine nanobody selection efforts, such as B. a time-extended and thus affinity-enhancing immunization scheme, subsequent affinity maturation including off-rate selections, and testing and improvement of approximately 200 initial candidates. When site-specifically labeled with fluorophores, the resulting nanobodies performed remarkably well in Western blotting and immunofluorescence. In contrast to polyclonal secondary antibodies, they even enable multicolored labeling and colocalization in one step.
UltraTek Anti-Mouse
ABJ008
ScyTek Laboratories
8 ml
96 EUR
UltraTek Anti-Mouse
ABJ015
ScyTek Laboratories
15 ml
117.6 EUR
UltraTek Anti-Mouse
ABJ125
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125 ml
273.6 EUR
UltraTek Anti-Mouse
ABJ500
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500 ml
664.8 EUR
UltraTek Anti-Mouse
ABJ999
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1000 ml
1120.8 EUR
UltraTek Anti-Rabbit
ABK008
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96 EUR
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15 ml
117.6 EUR
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ABK125
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125 ml
273.6 EUR
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500 ml
664.8 EUR
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1120.8 EUR
UltraTek Anti-Polyvalent
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117.6 EUR
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478.8 EUR
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500 ml
1444.8 EUR
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ABN999
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2439.6 EUR
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AGL008
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94.8 EUR
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AGL015
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115.2 EUR
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AGL125
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125 ml
249.6 EUR
UltraTek Anti-Goat
AGL250
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250 ml
427.2 EUR
UltraTek HRP Anti-Mouse Staining System
AFJ600
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500 Slides
340.8 EUR
UltraTek HRP Anti-Mouse Lab Pack
UHM125
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1250 Slides
482.4 EUR
UltraTek HRP Anti-Mouse Lab Pack
UHM500
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5000 Slides
1239.6 EUR
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UHM999
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10000 Slides
2106 EUR
Rabbit Polyclonal antibody Anti-CRBN
Anti-CRBN
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50 µg
418.8 EUR
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AFL600
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500 Slides
340.8 EUR
UltraTek HRP Anti-Mouse (DAB) Staining System
AKA080
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70 Slides
152.4 EUR
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AKB080
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152.4 EUR
UltraTek Alk-Phos Anti-Mouse Lab Pack
UAM125
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1250 Slides
572.4 EUR
UltraTek Alk-Phos Anti-Mouse Lab Pack
UAM500
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5000 Slides
1726.8 EUR
UltraTek Alk-Phos Anti-Mouse Lab Pack
UAM999
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10000 Slides
3174 EUR
UltraTek HRP Anti-Rabbit Staining System
AFK600
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500 Slides
340.8 EUR
UltraTek HRP Anti-Polyvalent Staining System
AFN600
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500 Slides
340.8 EUR
UltraTek HRP Anti-Polyvalent Lab Pack
UHP125
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1250 Slides
625.2 EUR
UltraTek HRP Anti-Polyvalent Lab Pack
UHP500
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5000 Slides
1532.4 EUR
UltraTek HRP Anti-Polyvalent Lab Pack
UHP999
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10000 Slides
2878.8 EUR
UltraTek HRP Anti-Rabbit Lab Pack
UHR125
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1250 Slides
482.4 EUR
UltraTek HRP Anti-Rabbit Lab Pack
UHR500
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5000 Slides
1239.6 EUR
UltraTek HRP Anti-Rabbit Lab Pack
UHR999
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10000 Slides
2106 EUR
UltraTek Horseradish Peroxidase
ABL008
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8 ml
96 EUR
UltraTek Horseradish Peroxidase
ABL015
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15 ml
117.6 EUR
UltraTek Horseradish Peroxidase
ABL125
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125 ml
273.6 EUR
UltraTek Horseradish Peroxidase
ABL500
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500 ml
664.8 EUR
UltraTek Horseradish Peroxidase
ABL999
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1000 ml
1120.8 EUR
UltraTek Alkaline Phosphatase
ABM008
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8 ml
130.8 EUR
UltraTek Alkaline Phosphatase
ABM015
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15 ml
177.6 EUR
UltraTek Alkaline Phosphatase
ABM125
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125 ml
504 EUR
UltraTek Alkaline Phosphatase
ABM500
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500 ml
1492.8 EUR
UltraTek Alkaline Phosphatase
ABM999
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1000 ml
2553.6 EUR
UltraTek Alk-Phos Anti-Mouse (Permanent Red) Staining System
AET080
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70 Slides
165.6 EUR
UltraTek Alk-Phos Anti-Rabbit Staining System
AFM600
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500 Slides
340.8 EUR
UltraTek Alk-Phos Anti-Polyvalent Staining System
AFP600
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500 Slides
340.8 EUR
UltraTek HRP Anti-Rabbit (DAB) Staining System
AKC080
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70 Slides
152.4 EUR
UltraTek HRP Anti-Rabbit (AEC) Staining System
AKD080
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70 Slides
152.4 EUR
UltraTek HRP Anti-Polyvalent (DAB) Staining System
AMF080
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70 Slides
165.6 EUR
UltraTek HRP Anti-Polyvalent (AEC) Staining System
AMG080
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70 Slides
165.6 EUR
UltraTek Alk-Phos Anti-Polyvalent Lab Pack
UAP125
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1250 Slides
860.4 EUR
UltraTek Alk-Phos Anti-Polyvalent Lab Pack
UAP500
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5000 Slides
2506.8 EUR
UltraTek Alk-Phos Anti-Polyvalent Lab Pack
UAP999
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10000 Slides
4620 EUR
UltraTek Alk-Phos Anti-Rabbit Lab Pack
UAR125
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1250 Slides
572.4 EUR
UltraTek Alk-Phos Anti-Rabbit Lab Pack
UAR500
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5000 Slides
1726.8 EUR
UltraTek Alk-Phos Anti-Rabbit Lab Pack
UAR999
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10000 Slides
3174 EUR
Polyclonal Goat anti-GST α-form
GST-ANTI-1
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50 uL
336 EUR
Polyclonal Goat anti-GST μ-form
GST-ANTI-2
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50 uL
336 EUR
Polyclonal Goat anti-GST p-form
GST-ANTI-3
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UltraTek Alk-Phos Anti-Rabbit (Permanent Red) Staining System
AME080
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70 Slides
152.4 EUR
UltraTek Alk-Phos Anti-Polyvalent (Permanent Red) Staining System
AMH080
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165.6 EUR
Mouse anti IgA1
10-I06A
Fitzgerald
1 mg
369.6 EUR
SensiTek Anti-Mouse
ABC008
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8 ml
85.2 EUR
SensiTek Anti-Mouse
ABC015
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15 ml
96 EUR
SensiTek Anti-Mouse
ABC125
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125 ml
211.2 EUR
SensiTek Anti-Mouse
ABC500
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500 ml
504 EUR
SensiTek Anti-Mouse
ABC999
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1000 ml
853.2 EUR
anti-mouse VLK
AR03-PA0002
Abfrontier
100 ul
400.8 EUR
Mouse Anti-FcεRIα
MOFCeRIaAP(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaAP(V25)
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25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaB(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaB(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaCFB(V100)
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100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaCFB(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaF(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaF(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaF(V500)
ImmunoStep
500 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPE(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPE(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPP(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPP(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPP55(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPP55(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPU(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-FcεRIα
MOFCeRIaPU(V500)
ImmunoStep
500 ug
60 EUR
Mouse Anti-IL2
MOIL2A(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-IL2
MOIL2A(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-IL2
MOIL2B(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-IL2
MOIL2B(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-IL2
MOIL2CFB(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-IL2
MOIL2CFB(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-IL2
MOIL2F(V100)
ImmunoStep
100 ug
60 EUR
Mouse Anti-IL2
MOIL2F(V25)
ImmunoStep
25 ug
60 EUR
Mouse Anti-IL2
MOIL2F(V500)
ImmunoStep
500 ug
60 EUR
Mouse Anti-IL2
MOIL2PE(V100)
ImmunoStep
100 ug
60 EUR
In stochastic optical reconstruction microscopy (STORM; Rust et al., 2006) of microtubules, an anti-mouse κ-light chain nanobody showed greatly reduced fluorophore offset distances, suggesting its use as a superior alternative to traditional anti-mouse secondary antibodies suggests. Furthermore, we show that anti-IgG nanobodies conjugated to HRP or expressed as fusions with ascorbate peroxidase (APEX2; Lam et al., 2015) and thus can be used for enhanced chemiluminescence Western blotting, colorimetric ELISAs, or immuno-EM detection be able. These monoclonal recombinant nanobodies are therefore a perfect replacement for conventional polyclonal secondary antibodies of animal origin. We imagine that they can be constructed in such a way