Figure 1. Western blot analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (MBS1753670).
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 50ug of sample under reducing conditions.
Lane 1: rat heart tissue lysates
Lane 2: rat lung tissue lysates
Lane 3: rat liver tissue lysates
Lane 4: human HEK293 whole cell lysates
Lane 5: human Jurkat whole cell lysates
Lane 6: human CACO-2 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-UBE2D1/2/3/4 antigen affinity purified polyclonal antibody (Catalog # MBS1753670) at 0. 5 μg/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 (Catalog # MBS176460) with Tanon 5200 system. A specific band was detected for UBE2D1/2/3/4 at approximately 17KD. The expected band size for UBE2D1/2/3/4 is at 17KD.
Figure 2. IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (MBS1753670).
UBE2D1/2/3/4 was detected in paraffin-embedded section of mouse lung lymph node tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8. 0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # MBS176451) with DAB as the chromogen.
Figure 3. IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (MBS1753670).
UBE2D1/2/3/4 was detected in paraffin-embedded section of human testicular cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8. 0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # MBS176451) with DAB as the chromogen.
Figure 4. IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (MBS1753670).
UBE2D1/2/3/4 was detected in paraffin-embedded section of human bladder cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8. 0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # MBS176451) with DAB as the chromogen.
Figure 5. IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (MBS1753670).
UBE2D1/2/3/4 was detected in paraffin-embedded section of rat lung tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8. 0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # MBS176451) with DAB as the chromogen.
Figure 6. IF analysis of UBE2D1/2/3/4 using anti- UBE2D1/2/3/4 antibody (MBS1753670).
UBE2D1/2/3/4 was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (MBS176582) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670) overnight at 4 degree C. DyLight®488 Conjugated Goat Anti-Rabbit IgG 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 7. Flow Cytometry analysis of A431 cells using anti-UBE2D1/2/3/4 antibody (MBS1753670).
Overlay histogram showing A431 cells stained with MBS1753670 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-UBE2D1/2/3/4 Antibody (MBS1753670, 1μg/1x106 cells) for 30 min at 20 degree C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x106 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
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