Epitope tag antibodies are very commonly used to study localization of a target protein in immunofluorescence (IF) assays. An epitope tag can either be fused at C- or N-terminus of a target gene and expressed in a desired expression system.

Note: Choice of vector and promoter system will depend on the expression system (mammalian, bacterial, etc.). Do consider the position of any functional or structural domains in the target protein before introducing an epitope tag. The tag should not interfere with target protein folding, function, or cellular localization.

Materials

Cell seeding and transfection for immunostaining

  1. Seed cells on either coverslips placed in culture plates or in chamber slides. A target gene fused to an epitope tag is cloned into an expression vector and transfected in the appropriate expression system as per standard protocols.
  1. After 48-72 hours of transfection, cells are processed for immunostaining.

Protocol tips

Remember to have untransfected, vector alone, and lipofectamine alone samples as controls when testing a construct for the first time.


Fixation of cells

  1. Remove and discard media from each well. Wash with 1X PBS for 5 minutes.
  1. Fixation using PFA or acetone/methanol:
    • PFA: Add 4% PFA (reconstituted in 1X PBS). Fix the cells for 10 minutes at room temperature. Remove PFA solution and wash with 1X PBS, 3 x 5 minutes.
    • Acetone/Methanol: Add ice-cold acetone/methanol. Place at -20°C for 5 minutes. Remove acetone/methanol and wash with 1X PBS, 3 x 5 minutes.
  1. Plates can be stored at 4°C up to 1 month in 1X PBS with 0.03% sodium azide.


Permeabilization and immunostaining of cells

  1. For permeabilization, add 0.1% Triton X-100 in PBS. Incubate for 15 minutes at room temperature.
  1. Remove Triton X-100 and wash with 1X PBS, 3 x 5 minutes. Block the cells using 2% BSA in 1X PBS with 0.03% sodium azide for 60 minutes at room temperature or plate can be stored at 4°C up to one month.
  1. For immunostaining, remove the blocking solution (do not wash the cells at this point) and add the epitope tag antibody (2 to 5 µg/mL) in 0.1% BSA in 1X PBS. Incubate for 3 hours at room temperature or overnight at 4°C.
  1. Wash with 1 mL of 1X PBST, 3 x 5 minutes.
  1. Incubate with recommended dilution of fluorophore-conjugated secondary antibody along with labelled phalloidin prepared in 0.1% BSA and incubate for 60 minutes at room temperature in the dark.
  1. Wash with 1 mL of 1X PBST, 3 x 5 minutes.

Protocol tips

Equal concentrations of epitope tag antibody should be added to untransfected/transfected samples and any other controls.

For co-localization studies:

  • If you have an antibody against your target protein available, you could also add it along with epitope tag antibody to confirm co-localization between the endogenous and overexpressed epitope-tagged protein.
  • Ensure that the host species of the tag and target antibodies are different. This will avoid any cross-reactivity.
  • Choose secondary antibodies with different fluorophores such that their spectral ranges do not overlap.
     

For primary conjugates:

No secondary antibody is needed. If you are using a fluorophore-conjugated epitope tag antibody, it is extremely important to have an isotype control. For IF, an isotype control would be a host-matched isotype conjugated with the same fluorophore as the tag antibody. This will help you assess the background signal.


Slide preparation for imaging

  1. If cells are grown on coverslips, wipe the glass slides to be used for mounting with 70% ethanol and allow them to dry. For chamber slides, separate the chamber using the chamber slide remover.
  1. Mounting coverslips:
    • Apply 10 µL of ProLong Diamond Antifade Mountant with DAPI on the surface of the glass slides. Then, carefully remove the coverslips containing the cells from the well and mount it upside down onto the DAPI solution. Blot the excess solvent from non-sample surface using a soft tissue.
  • Mounting chamber slides:
    • Add 10 to 20 µL of DAPI to each well of the chamber slide and mount it with coverslip by placing it on top of the chamber slide.
  1. Place the slides in the dark for 24 hours at room temperature. Then, image using a fluorescence microscope.

Protocol tips

Capture images of untransfected, transfected, no primary/isotype controls at the same exposure settings to compare signal/noise ratios.

Resources

For Research Use Only. Not for use in diagnostic procedures.