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Thank you for the information. I will attempt to use without conjugation initially.
Is there a protocol that you can recommend when using these antibodies and should the cells be permeabilised as the GLUT4 is translocated to the plasma membrane?
Thanks in advance,
Asked on Apr 20 2012
We used the following protocol with Anti-Glucose Transporter GLUT4 antibody (ab62375):
NSO cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab62375, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (https://www.abcam.com/Goat-Rabbit-IgG-HL-DyLight-488-preadsorbed-ab96899.html) at 1/500 dilution for 30 min at 22°C.
Isotype control antibody was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
This antibody also gave a positive signal in NSO cells fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% PBS-Tween used under the same conditions.
For both of these methods permiabilisation was used.
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Answered on Apr 20 2012