Recently, i had cloned my GOI along with flag tag in a retroviral construct and after transfection in 293T cells i could detect my protein using anti FLAG antibody from Santa Cruz but when i tried to probe it with anti- Flag antibody from other companies (Sigma and few others) i wasn't able to detect my band but was able to detect flag expression in positive control, i am not able to understand why my construct is been detected by a single antibody and why not others?
Hi Yashika,
could you please describe in more details your experiment? Could you also rule out the possibility that the first antibody give a false positive?
Best,
Piero
Hi there,
Have you run both WB at the same time with the same samples? If not, FLAG tagged protein might have run off between the 2 experiments. If yes, there might be a problem of sensitivity in the detection process (does the positive control give similar signal with both detections?).
Hey, Piero,
I have cloned my gene along with C-ter FLAG in a retroviral construct. After transfection, i am getting FLAG expression at the desired position. With the same extracts if i probe with endogenous antibody i get expression at the same position ruling out the possibility of False positive. But when i try with FLAG antibody from different company i doesn't work. But ya i do get expression in positive control with these antibodies.
Hey Dominique,
I have done western with the same extracts but not at the same time, but my positive control gives FLAG expression with other antibodies as well.
Is there a possibility that 2x FLAG is more sensitive as compared to 1x FLAG?
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