3X HA Tag (Triple-HA Tag) is a bioactive peptide consisting of three repetitive HA tags (YPYDVPDYA) linked by peptide bonds and is mainly used for protein and peptide detection as well as facilitating functional analysis of target proteins. 3X HA Tag can be specifically recognized and conjugated to anti-HA tag antibody, and is widely used in Western blot, immunofluorescence, immunoprecipitation and other experimental techniques.

Western blot and immunofluorescence detection of fusion proteins
I. Inoculation of cells
1. Add a total of 1.5 × 10^4 cells to each well of a 24-well plate.
2. Move to a 37 °C incubator overnight.
II. Transfection of HeLa cells using JetPrime
1. Remove the medium from each well and replace with 0.5 ml complete DMEM.
2. Add 500 ng pCDNA3-VAMP8-3xHA and adjust to a final volume of 100 μl with JetPrime buffer.
3. Incubate at room temperature for 10 -15 minutes.
4. Add 15 μl of transfection mixture to each well.
5. Incubate in a tissue culture incubator at 37 °C for 6 hours.
6. Remove the medium and replace with 1.5 ml complete DMEM.
7. Incubate overnight in a 37 °C tissue culture incubator.
III. VAMP8 endocytic uptake assay
1. Wash transfected cells with 1 ml ice-cold DMEM. Repeat once more for a total of two washes.
2. Dilute rabbit anti-HA antibody in ice-cold complete DMEM to a final dilution of 1:200.
Add 150 μl of diluted anti-HA antibody to each well.
3. Incubate on ice for 60 min with occasional shaking.
4. Wash cells 3 times with 1 ml ice-cold complete DMEM.
5. Wash cells twice with 1 ml pre-warmed EBSS (37 °C).
6. Add 1 ml pre-warmed EBSS to each well and incubate in a 37 °C tissue culture incubator for 15, 45, and 90 min.
III. Immunofluorescence
1. At each time point, remove the appropriate coverslip and transfer to a new well of a new 24-well plate containing 1 ml 1× PBS.
2. Remove the 1× PBS and replace with 1 ml 4% paraformaldehyde solution.
3. Incubate at room temperature for 15 minutes.
4. Remove the 4% paraformaldehyde solution and wash 3 times with 1 ml 1× PBS. Incubate at room temperature for 5 minutes for each wash.
5. Remove the last wash and block in 1 ml blocking buffer for 1 hour at room temperature.
6. Dilute mouse anti-Lamp1 or mouse anti-EEA1 to a final dilution of 1:100 or 1:1,000, respectively, in antibody incubation buffer.
7. Remove the blocking buffer, replace with 100 μl of the appropriate diluted antibody, and incubate at 4 °C.
8. Remove the diluted antibody solution and wash three times with 1 ml 1× PBS. Incubate at room temperature for 5 minutes for each wash.
9. Dilute Alexa-conjugated secondary antibodies. Dilute goat anti-mouse Alexa-488 and goat anti-rabbit Alexa-546 to a final dilution of 1:250 in antibody dilution buffer.
10. Remove the wash solution, add 200 μl of the diluted secondary antibody solution, and incubate at room temperature for 1 hour in the dark to minimize photobleaching.
11. Remove the solution containing secondary antibody and wash three times with 1 ml 1× PBS. Incubate at room temperature for 5 minutes for each wash.
12. Add 200 μl of 1× PBS containing 1 μg/ml DAPI and incubate at room temperature for 5 minutes.
13. Remove the solution containing DAPI and wash twice with 1 ml 1× PBS. After each wash, incubate at room temperature for 5 minutes.
14. Pipette 10 μl of FluorSave mounting medium onto a microscope slide.
15. Image on an appropriate confocal or widefield microscope system. [2]