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Cant seem to detect HA-tagged protein with immunofluorescence after transfection

fitc HA-tag

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#1 KhaleesiDany

KhaleesiDany

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Posted 10 November 2015 - 10:02 AM

Greetings

 

I generated a plasmid to encode for a protein, the plasmid includes a HA-tag for detection, my protein also has a nuclear localization signal. 48 hours post transfection i do immunofluorescence staining however i only pick up fitc signal in the cytoplasm and not in the nucleus. we think the problem might be that we not geting adequate permeabilization of the nuclear membrane only the cell membrane.And yes i know it is going to the nucleus because it is a DNA binding protein and my other assays of activity show that the protein is active which means it is going in to the nucleus however we can only pick up the ones in the cytosol. has any one detected anything in a nucleus before? if yes how. or if anyone has suggestions on what i could change. Thanks  below is the protocol i use: 

Immunofluoresence (24 well plate protocol) Fixing and Permeablizing: 1. Make 4% PFA: Dissolve Paraformaldehyde in PBS and incubate at 70°C with occasional vortexing to dissolve 2. Aspirate off media from cells and wash with 500 µl 1xPBS

3. Aspirate off PBS and add 500 µl 4% PFA to fix cells. Incubate RT for 15 minutes.

4. Wash 2x with 500 µl 1x PBS

5. Aspirate off PBS and add 500 µl 0.1% Triton-X in PBS to permeablize cell membrane. Incubate RT for 10 minutes

6. Wash 2x with 500 µl 1x PBS (can store at 4°C or proceed) Immunostaining:

7. Block 1 hour in 25 µl 1% BSA/PBS in humidity chamber (Petri plate, damp paper towel, parafilm)

8. Incubate in 25 µl 1° Ab (usually 1:200 in 1%BSA/PBS) for 1 hour in humidity chamber

9. Wash 2x with 500 µl 1x PBS

10.Incubate in 25 µl 2°Ab (1:200 in 1% BSA/PBS) for 40 minutes in humidity chamber. Place in dark so as to not bleach fluorescent 2° Ab

11.Wash 2x with 500 µl 1x PBS

12.Mount on slides with DAPI mounting media and seal with clear nail polish

 

thanks







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