SouthernBiotech
Goat anti-mouse IgG(H+L), human ads-FITC
1031-02
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In our research, we need to examine the binding of antibodies to their corresponding cell surface receptor. In addition, we need to examine the immunoreactivity of our antibody to demonstrate that chelation did not negatively affect the binding of the antibody. Our protocol for this procedure requires the use of a fluorescent-labeled secondary antibody. We chose to use this antibody because we thought that any FITC-labeled goat anti-mouse antibody would work for our project.
Flow Cytometry
Human lung cancer cells
Cells were incubated with either PBS, 5 μg/mL of YY146, 25 μg/mL of YY146, 5 μg/mL of NOTA-YY146, or 25 μg/mL of NOTA-YY146 for 30 min at 25-30 °C.
There was no blocking agent used in this study.
Cells were incubated with the FITC-labeled goat anti-mouse secondary antibody (5 μg/mL; Southern Biotech) for 30 min at 25-30 °C.
There was no tertiary incubation. Cells were washed 3-times with PBS before detection.
The binding efficiency was analyzed using a MACSQuant cytometer (Miltenyi Biotech) and mean fluorescence intensities were processed using the FlowJo analysis software (Tree Star, Inc.).
H460 and H23 cells displayed enhanced fluorescence signal when incubated with both YY146 and NOTA-YY146 suggesting high levels of CD146 expression in these two cell lines. There were no appreciable differences in the binding affinity of NOTA-YY146 or YY146 alone indicating that NOTA-conjugation did not affect the immunoreactivity of YY146 to CD146. A549 and H4006 cells showed similar results to H460 and H23, yet at a lesser degree which suggests lower levels of CD146 expression. H358 cells showed a slight increase in fluorescence signal when incubated with 25 µg/mL of YY146 or NOTA-YY146, yet the lower concentration of YY146 or NOTA-YY146 (5 µg/mL) failed to provide a signal difference. This suggests that H358 cells express CD146 at low levels. Lastly, H522 cells did not display an increase in fluorescence signal when incubated with YY146 or NOTA-YY146, indicating that this cell line may lack CD146.
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Fast shipping, ease of use.
Does not work correct all the time, needs significant optimization
There are better antibodies on the market, but this antibody will work with some good optimization.