Scientific Forum

Western blot of ENaC protein

Hi, BiCell,

I believe the new purification method worked. In the blot below, we transfected HEK cells with mouse ENaC (alpha subunit has a V5 tag at the C-terminus).
We included a negative control (plain HEK cell lysates) and a positive control (alpha pulldown by V5 antibody). We can see that the purified #65 recognizes a specific
band in the HEK+ ENaC lysates, which is the same size as the V5 IP and the correct size for ENaC alpha.
This is really exciting, but we would like to try if the antibody can recognize ENaC alpha in the native tissues. We will update you with the new data.

Best, Shujie

Comments

  • September 30, 2023

    bicell-admin

    Hi BiCell,

    I received the antisera and tested them with WB. One of them #65 looks promising. The expected bands are marked with green arrows. These bands are weak but specifically detected only in cells transfected with mouse ENaC.
    I suggest we focus on this clone in the next step of purification. Let me know your thoughts.

    Thanks,
    Shujie

  • September 30, 2023

    bicell-admin

    Hi Dr Shi,

    We have completed 2nd round of purification that has isolated the fraction specific for unfolded ENaC protein. We will ship you antibody for delivery on Thursday.

    PS. We would recommend to remove glycerol from the lysis buffer because there are 2 detergents present.

    Regards,
    BiCell Team

  • September 30, 2023

    bicell-admin

    Dear Dr Shi,

    Your custom rabbit pAb project is complete. Please review the attached progress report with ELISA data.

    Both animals reached titer of 1M. Both will give great polyclonal antibodies. If you are interested, we can take your project to rabbit monoclonal selection.

    We will ship purified IgG to you within 5 business days. Please test them and let us know if they work so we can supply you with 1mg of IgG.

    Best regards
    BiCell Scientific Team

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