Mouse anti v5 tag monoclonal antibody

Infected cells grown on glass coverslips were fixed and permeabilized using 100% cold methanol for 10 min. These were then washed 3 times with PBS for 5 min and blocked using 3% (wt/vol) bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT). HA was detected with rat monoclonal anti-HA antibody 3F10 (Roche), SAG2A was detected using rabbit polyclonal anti-SAG2A antibodies (55 (link)), V5 was detected with mouse anti-V5 tag monoclonal antibody (Invitrogen R960), human CC2D1A was detected with rabbit anti-CC2D1A antibodies (Sigma HPA005436), human PDCD6IP/ALIX was detected with rabbit anti-ALIX antibodies (a gift from Wesley Sundquist, University of Utah), and biotinylated proteins were detected with streptavidin Alexa-Fluor-488 (Invitrogen S32354). Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Primary and secondary antibodies were both diluted in 3% BSA in PBS. Coverslips were incubated with primary antibodies for 1 h at RT, washed, and incubated with Alexa-Fluor-conjugated secondary antibodies (Invitrogen) for 1 h at RT. Vectashield with DAPI stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using wide-field epifluorescence microscopy, and images were analyzed using ImageJ/FIJI software (NIH).

Monolayers of infected cell on glass coverslips were fixed with cold methanol for 12 min. Samples were washed with PBS and blocked using 3% bovine serum albumin (BSA) in PBS for at least 30 min. SAG1 was detected with rabbit anti-SAG1 polyclonal antibody and V5 was detected with mouse anti-V5 tag monoclonal antibody (Invitrogen). Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Primary and secondary antibodies were both diluted in 3% BSA in PBS. Coverslips were incubated with primary antibodies for 30 min, washed, and incubated with secondary antibodies for 30 min. Vectashield with DAPI stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using wide-field epifluorescence microscopy and images were analyzed using ImageJ. All images shown for any given condition/staining in any given comparison/dataset were obtained using identical parameters.

Cell lysates were prepared at the indicated time points postinfection in Laemmli sample buffer (Bio-Rad) supplemented with 355 mM 2-mercaptoethanol (BME). The samples were boiled for 5 min, separated on a Bolt 4 to 12% Bis-Tris gel (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS) supplemented with 0.5% Tween 20, and proteins were detected by incubation with primary antibodies diluted in blocking buffer followed by incubation with secondary antibodies (raised in goat against the appropriate species) conjugated to horseradish peroxidase (HRP) and diluted in blocking buffer. HA was detected using an HRP-conjugated HA antibody (Roche 12013819001), V5 was detected with mouse anti-V5 tag monoclonal antibody (Invitrogen R960), SAG2A was detected using rabbit polyclonal anti-SAG2A antibodies (55 (link)), MAF1b was detected using rabbit polyclonal anti-MAF1b antibodies (27 (link)), GAPDH was detected using mouse monoclonal anti-GAPDH antibody 6C5 (Calbiochem), and biotinylated proteins were detected using streptavidin-HRP (Invitrogen S911). HRP was detected using an enhanced chemiluminescence (ECL) kit (Pierce). Silver-stained gels were generated using the Pierce silver stain kit (Thermo Scientific).