Expedeon’s Conjugate Check&Go! Kit allows scientists to confirm the success of their antibody conjugation in one easy step.
The key component of the kit is a nitrocellulose membrane containing a ‘Test line’ of immobilized Protein A and Protein G called a “half strip”.
Both Protein A and Protein G have a high affinity for the Fc region of a variety of IgG molecules. The “half strips” also contain an absorbent pad to promote and control the flow of sample through the nitrocellulose. This simple qualitative lateral flow assay does not require any specialized or costly equipment.
*Please note Conjugate Check&Go! is not compatible with the following Lightning-Link® fluorescent labels: AMCA, Atto350, Atto390, Dylight® 405, Dylight® 800, Dylight® 755, Dylight® 700 and Dylight® 680.
Schematic representation of the Conjugate Check&Go! process
When the antibody conjugate is run on the Conjugate Check&Go! strip, it flows along the nitrocellulose, binding to the Protein A and Protein G concentrated on the Test line. When the antibody is successfully conjugated to a colored label, a visible line appears on the strip.
Checking Your InnovaCoat® GOLD Conjugation
Gold conjugate concentration is expressed in Optical Density (OD); for instance, a mini vial of InnovaCoat® GOLD conjugation kit contains 50ul of 20 OD gold conjugate. The OD can be checked at specific wavelengths according to the gold nanoparticle size, using an UV-vis spectrophotometer.
Our Conjugate Check&Go! strips allow the researcher to visualize InnovaCoat® GOLD conjugates at different ODs. In addition, gold conjugates made from different sources of gold can be tested on the strips (i.e. passively conjugated gold nanoparticles and other nanogold conjugation kits). The same OD range applies according to the gold nanoparticle size.
InnovaCoat® GOLD Conjugation Results Using Check&Go! Strips:
Figures 1–4. Rabbit IgG has been conjugated to 10, 20, 40 or 80nm InnovaCoat® GOLD and tested on Conjugate Check&Go! strips at different concentrations. A concentration range is suggested on the Conjugate Check&Go! protocol according to the size of the gold nanoparticles tested. Gold conjugates prepared with 10nm and 80nm InnovaCoat® GOLD (Fig. 1 and 4) require a higher concentration than 20nm and 40nm InnovaCoat® GOLD (Fig. 2 and 3) to produce an intense red line on the strip.
Checking Your Latex Conjugation
The concentration of latex conjugates is expressed in % solids (w / v); for instance, a mini vial of Expedeon’s Latex Conjugation kit contains 40ul of 1% latex conjugate. Our Conjugate Check&Go! strips allow the researcher to visualize latex conjugates at different concentrations of latex.
All our latex beads, Blue, Black and Red, have been tested using our Conjugate Check&Go! (see Figure 5. below). In addition, conjugates generated from different sources of latex can be tested on the strips (the same concentration range applies). We recommend to use latex–conjugates in a range comprised between 0.02–0.004%.
Latex Conjugation Results Using Check&Go Strips:
Figure 5. Rabbit IgG has been conjugated to 400nm Latex beads and tested at different concentrations. The Conjugate Check&Go! strips have been pre-treated with the running buffer provided plus 1% of Bovine Serum Albumin (BSA) to prevent unspecific binding on the T Line.
Checking Your Lightning-Link® Conjugation
The concentration of conjugates produced using Lightning-Link® reflects the starting concentration of the antibody. The Conjugate Check&Go! strips allow visualization of conjugates generated using our Lightning-Link® fluorescent dyes and proteins at different concentrations of antibody. However, enzymes cannot be detected on these strips. Antibody conjugates produced using other antibody labeling technologies can also be tested as long as the colored label is bright enough to be visible on the strips.
Lightning-Link® Conjugation Results Using Check&Go! Strips:
Figures 6–7. Fluorescent dyes. Rabbit IgG has been conjugated to Lightning-Link® Fluorescein and Lightning-Link® Rapid Cy5.5 and tested on Conjugate Check&Go! strips at different concentrations. The suggested range for fluorescent dye conjugated antibodies is 100–1,000ug / ml. The intensity of the line also depends on the color of the label being used. 10μg Lightning Link® kits may not be suitable for some dilutions.
Figure 8. Fluorescent proteins. Rabbit IgG has been conjugated to Lightning-Link® RPE and tested on Conjugate Check&Go! strips at different concentrations. The suggested range for fluorescent protein conjugated antibodies is 5–100ug / ml. The intensity of the line also depends on the color of the label being used.
Figure 9. Fluorescent tandems. Rabbit IgG has been conjugated to Lightning-Link® APC/Cy5.5 and tested on Conjugate Check&Go! strips at different concentrations. The suggested range for fluorescent protein conjugated antibodies is 50–1,000ug / ml. The intensity of the line also depends on the color of the label being used.
1. What is the best storage condition for the kit?
The kit is shipped at ambient temperature and its components need different storage temperature. 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-GOLD conjugate is stable at 4⁰C for up to 2 weeks in the 1x Running Buffer + BSA. For long term storage, reconstitute the control vial in 40ul 1x Running Buffer (without BSA) and dilute further in 1x Running Buffer + BSA to run the LFA. Keep the 10x Running Buffer at 4⁰C.
2. Can antibodies from different species be used?
Yes, the Conjugate Check&Go! 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.
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 used the Conjugate Check&Go! Kit to confirm successful conjugation of my antibody to any Lightning-Link label?
The Conjugate Check&Go kit is a quick immunochromatographic test designed to detect on the T line IgGs conjugated labels visible by eye, therefore the following labels are not compatibale with this kit: AMCA, Atto350, Atto390, Dylight® 405, Dylight® 800, Dylight® 755, Dylight® 700 and Dylight® 680, horseradish peroxidase, alkaline phosphatase, streptavidin or biotin.
5. How can I check my antibody conjugates if the label has a faint color?
If your antibody is conjugated to a faint label that is hardly detectable by eye, you can run your conjugate at higher concentration than the one recommended to obtain a visible line on the strips.
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