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Biotin Check&Go!

Biotin Check&Go! allows scientists to confirm the success of their antibody biotinylation in one easy step.

Unlike traditional methods of assessing the success of biotinylation, such as the HABA assay, which can be tedious and time consuming, Biotin Check&Go! is a simple immunochromatography test that requires only 10 minutes of your time and very small quantities of your antibody conjugate.

The key components of the kit include a nitrocellulose membrane containing a Test line of immobilized Protein A and Protein G, both of which have a high affinity for the Fc region of a variety of IgG molecules, and a 40nm InnovaCoat® GOLD Streptavidin Conjugate for detection.

  • Quickly check the success of your conjugation (only 10 minutes!)
  • Easy to use
  • No costly equipment required
  • Compatible with antibody conjugates generated using our Lightning-Link® Biotin Antibody Labeling Kits and other biotin labeling technologies available commercially
  • Requires only small volumes (20ul of diluted conjugate)
  • Unique to Expedeon.

When the biotinylated antibody is mixed with the streptavidin–gold conjugate it forms a complex that flows along the nitrocellulose membrane. On binding to the Protein A and Protein G concentrated on the Test line, a visible red line appears on the strip.

 

Figure 1. Rabbit IgG has been conjugated with Lightning-Link® Rapid Biotin A and run on Protein A / G strips at different concentrations, together with 0.4OD InnovaCoat® GOLD Streptavidin. The intensity of the T line is proportional to the concentration of the biotinylated IgGs. A red line appears even at low IgGs concentration.

 

Figure 2. Polyclonal antibodies from rabbit, goat, mouse and rat have been conjugated with Lightning-Link® Rapid Biotin A and run on Protein A / G strips at different concentrations, together with 0.4OD InnovaCoat® GOLD Streptavidin. Antibodies from all species provided a red visible line on the strips.

1. What is the best storage condition for the kit?

The kit is shipped at ambient temperature and its components need different storage temperatures. Upon arrival, the plastic pot containing the strips, manufactured with molecular sieves integrated into the walls, must be kept at 4⁰C. The lyophilized control vial must be stored at -20⁰C; once reconstituted, the Rabbit IgG-biotin conjugate is stable at 4⁰C for up to 1 year in the 1x Running Buffer + BSA. The lyophilized 40nm InnovaCoat® GOLD Streptavidin must be stored at -20⁰C; once reconstituted it is stable at 4⁰C for up to 2 weeks. Keep the 10x Running Buffer at 4⁰C.

2. Can antibodies from different species be used?

Yes, the Protein A/G strips have been tested with antibodies from a variety of species including mouse, rabbit, goat and rat, providing they are conjugated to a colored label. The binding properties of Protein A and G, immobilized on the Test line of the strips, differ among species and subclasses of IgG. A low or no affinity for a specific IgG subclass may lead to low/no signal but the conjugate may be still fine. Affinity of Protein A and G for different Ig subclasses is reported in the table below.

SpeciesIg sub classesBinding to Protein ABinding to Protein G
RabbitIgGHighHigh
HumanIgG1HighHigh
IgG2HighHigh
IgG3No affinityHigh
IgG4HighHigh
IgALowNo affinity
IgDLowNo affinity
IgELowNo affinity
IgMLowNo affinity
PigIgGHighHigh
MouseIgG1Low/MediumMedium
IgG2aHighHigh
IgG2bHighMedium
IgG3Low/MediumMedium
IgMLowNo affinity
GoatIgGLowHigh
SheepIgGLowHigh
RatIgGLowHigh
IgG1LowLow/Medium
IgG2aLowHigh
IgG2bLowLow/Medium
IgG2cLowLow/Medium
IgMLowNo affinity

3. Can conjugates from antibody fragments be checked?

The protein A and protein G immobilized on the Test line have a high affinity for the Fc region of IgG molecules, and therefore for the Fc fragments as well.

4. Can I use the Biotin Check&Go! kit to confirm successful conjugation of my antibody to other labels?

Biotin Check&Go! can only be used to check whether or not your biotinylation has been successful however we do provide another kit that enables the detection of antibody conjugation to fluorescent dyes and proteins, latex beads and gold nanoparticles. Find out more about this product here.

5. Can I use the Biotin Check&Go! kit to quantify the level of biotinylation?

No, you can’t. Biotin Check&Go! is a rapid qualitative method. However, you can discriminate different degrees of biotinylation based on the line intensity of your diluted conjugate.

Please see here for ordering & shipping information.

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