Flow cytometry was performed on a Navios flow cytometer (Beckman Coulter) as described earlier [30 (link)]. For binding studies, the immunoligands were applied at a concentration of 50 μg/ml. An Alexa Fluor 488-coupled anti-penta His secondary antibody (Qiagen) was employed for detection. To demonstrate simultaneous binding, SK-BR-3 cells were reacted with the immunoligands at 50 μg/ml and then incubated with an Fc-fusion protein containing the corresponding NK cell receptor (i.e. NKp30-Fc, NKp80-Fc, or DNAM-1-Fc) at 100 μ g/ml and subsequently with polyclonal FITC-coupled anti-human IgG F(ab’)2 fragments (Beckman Coulter). Receptor expression levels were quantitated by determination of specific antigen binding capacities (SABC) employing Qifikit (DAKO) according to manufacturer's protocols.
Alexa fluor 488 coupled anti penta his secondary antibody
Manufactured by Qiagen
The Alexa Fluor 488-coupled anti-penta His secondary antibody is a laboratory reagent used to detect and visualize proteins with a poly-histidine (His) tag. It consists of a secondary antibody labeled with the Alexa Fluor 488 fluorescent dye, which binds to the His tag and emits a green fluorescent signal upon excitation. This allows for the identification and localization of His-tagged proteins in various analytical techniques.
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Lab products found in correlation
2 protocols using alexa fluor 488 coupled anti penta his secondary antibody
1
Flow Cytometry Analysis of Immunoligand Binding
2
ULBP2:HER2 Immunoligand Binding Assay
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Flow cytometry was performed on a Navios flow cytometer (Beckman Coulter) as described previously (Kellner et al. 2012b) . To analyze specific binding, ULBP2:HER2-scFv or the control immunoligand were employed at a concentration of 50 μg/mL. An Alexa Fluor 488-coupled anti-penta His secondary antibody (Qiagen) was used for detection. To analyze simultaneous antigen binding abilities, SK-BR-3 cells were incubated with the immunoligands at 50 μg/mL and then reacted with NKG2D-Fc (100 μg/mL), which was subsequently detected with polyclonal FITC-coupled anti-human IgG F(ab′) 2 fragments (Beckman Coulter). Cell surface ULBP2 was analyzed with a PE-conjugated anti-ULBP-2/5/6 (clone 165903; R&D Systems) antibody. A recommended corresponding isotype antibody was used as a control.
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