3X HA Tag

1. 3X HA Tag was amplified from pMPY-3HA plasmid series^[1]. The pMPY-3xHA was constructed by ligating a NotI 3xHA fragment into the NotI site of pBluescript 11 SK-, yielding plasmid pGTEP1. A SmaI URA3 fragment from pJJ242 was blunt-end ligated into the SmaI site of pBluescript II SK- in both orientations to create vectors pWS8931 and pWS8932. An XbaI-EcoRI URA3 fragment of pWS8931 was ligated into the XbaI EcoRI cut pGTEPl to create pGTEP1 -URA3. Primers HA-up 5’-ACGATCGTCGAATTCGA GCTCATCTTTTACCCATACGAT-3’ and HA-down 5’-TTCGACTGACTCGAGACTAGTAGC GTAATCTGGAACGTC-3’ were used to amplify the 3x HA epitope. This PCR fragment was digested with EcoRI and XhoI and ligated to the similarly restricted pGTEPl -URA3 vector to create the final product, pMPY-3xHA.
2. 3xHA Tag can be used for western blot and immunofluorescence detection of fusion protein^[2].
(1) On day1, Add a total of 1.5 x 10⁴ Hela cells per well of a 24-well plate. Bring to 1.5 mL of complete DMEM per well and move to 37°C incubator overnight (o/n).
(2) On day2, remove media from each well and replace with 0.5 mL of complete DMEM. Transfect 500 ng of pCDNA3-VAMP8-3xHA into Hela cells. Incubate 6 hours at 37°C in the tissue culture incubator. Remove the media and replace with 1.5 mL of complete DMEM. Incubate overnight in the 37°C tissue culture incubator.
(3) On day 3, wash transfected cells with 1 mL of ice-cold DMEM for two washes. Remove all the media. 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. Incubate on ice for 60 min. Wash cells three times with 1mL of ice-cold complete DMEM. Wash cells two times with 1 mL of pre-warmed EBSS (37°C). Add 1 mL of pre-warmed EBSS to each well, and incubate in the 37°C tissue culture incubator for 15, 45 and 90 min.
(4) At each time point, remove the appropriate coverglass and transfer into a new well of a new 24-well plate containing 1 mL of 1x PBS. Remove the 1x PBS, and replace with 1 mL of the 4% paraformaldehyde solution. Incubate for 15 min at room temperature. Remove 4% paraformaldehyde solution, and wash three times with 1 mL of 1x PBS. For each wash, incubate 5 min at room temperature. Remove last wash and block in 1 mL of blocking buffer for 1 h at room temperature. Dilute mouse anti-Lamp1 or mouse anti-EEA1 to final dilutions of 1:100 or 1:1,000, respectively, in antibody incubation buffer. Remove blocking buffer and replace with 100 μL of the appropriately diluted antibody, and incubate at 4°C o/n. Remove diluted antibody solution, and wash 3 times with 1 mL of 1x PBS. For each wash, incubate 5 min at room temperature. Dilute Alexa-coupled 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. Remove wash solution, add 200 μL of diluted secondary antibody solution, and incubate 1 h at room temperature in the dark to minimize photobleaching. Remove secondary antibody-containing solution, and wash three times with 1 mL of 1x PBS. For each wash, incubate 5 min at room temperature. Add 200 μL of 1x PBS containing 1 μg/mL of DAPI and incubate 5 min at room temperature. Remove DAPI-containing solution and wash twice with 1 mL of 1x PBS. For each wash, incubate 5 min at room temperature. Pipet 10 μL of FluorSave mounting media on a microscope slide. Remove coverglass from individual wells using forceps, and gently remove as much liquid as possible. Gently drop the coverglass onto the slide and the mounting media, making sure that no air bubbles are trapped between the coverglass and the slide. Remove extra mounting solution, and let the slide cure o/n at 4 °C. Image on an appropriate confocal or widefield microscopy system.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.