Hello Melani Juric

Besides ELISA, you may use Western Blot or Dot Blot. Dot Blot is a simpler, quicker version of Western Blot where you spot the sample directly onto the membrane without electrophoresis, which is then blocked to prevent non-specific binding. After blocking, antibodies are used to bind to the target molecule within the spots. Finally, the spots are visualized, often through chemiluminescence or fluorescence.

If you are planning to use ELISA, some important points you may want to consider while performing ELISA.

a. If your secreted protein is low in abundance, you may have to concentrate the supernatant. Choose an ultrafiltration spin column with a Molecular Weight Cutoff (MWCO) at least two times smaller than the molecular weight of your HA-tagged protein. For instance, if your protein is 30kDa, you may use a 10 kDa MWCO filter.

b. Always include a negative control, such as supernatant from untransfected cells to ensure that you are not detecting non-specific background proteins.

c. Be aware that the HA-tag can be cleaved by caspases during apoptosis, so avoid using supernatant from apoptotic or stressed cells.

d. Does your protein construct include a signal peptide to direct it into the secretory pathway, otherwise it will remain intracellular?

You may want to refer to the kit that is directed towards the human HA-tag as provided in the link below.

https://www.mybiosource.com/human-elisa-kits/hatag/7273505#:~:text=MBS7273505%20is%20a%20ready%2Dto,a%20substrate%20for%20HRP%20enzyme.

Best,

@ S.BRINDHA