Figure 1. Western blot analysis of USP5 using anti-USP5 antibody (MBS1755373).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human A549 whole cell lysates,
Lane 2: human U20S whole cell lysates,
Lane 3: human MDA-MB-453 whole cell lysates,
Lane 4: huamn MCF-7 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat testis tissue lysates,
Lane 7: mouse brain tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-USP5 antigen affinity purified polyclonal antibody (#MBS1755373) at 0.25 ug/mL overnight at 4 degree C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for USP5 at approximately 96 kDa. The expected band size for USP5 is at 96 kDa.
Figure 2. IF analysis of USP5 using anti-USP5 antibody (MBS1755373).
USP5 was detected in an immunocytochemical section of A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/mL rabbit anti-USP5 Antibody (MBS1755373) overnight at 4 degree C. DyLight488 Conjugated Goat Anti-Rabbit IgG (MBS177405) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37 degree C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 3. Flow Cytometry analysis of U251 cells using anti-USP5 antibody (MBS1755373).
Overlay histogram showing U251 cells stained with MBS1755373 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-USP5 Antibody (MBS1755373, 1 ug/1x10^6 cells) for 30 min at 20 degree C. DyLight488 conjugated goat anti-rabbit IgG (MBS177405, 5-10 ug/1x10^6 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1 ug/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
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