UBE2D1/2/3/4 Antibody

• Catalog Number: orb692216
• Category: Antibodies
• Description: UBE2D1/2/3/4 Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived human UBE2D1/2/3/4 recombinant protein (Position: D116-M147).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.25-0.5μg/ml, Human, Rat Immunohistochemistry (Paraffin-embedded Section), 2-5μg/ml, Human, Mouse, Rat Immunocytochemistry/Immunofluorescence, 5μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human, Mouse, Rat Direct ELISA, 0.1-0.5μg/ml, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 17 kDa
• UniProt ID: P51668
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500μg/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). 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 # orb692216) at 0.5 μg/mL overnight at 4°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 # orb90503) 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.

IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). 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 (orb692216) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). 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 (orb692216) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). 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 (orb692216) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). 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 (orb692216) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IF analysis of UBE2D1/2/3/4 using anti-UBE2D1/2/3/4 antibody (orb692216). UBE2D1/2/3/4 was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-UBE2D1/2/3/4 Antibody (orb692216) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of A431 cells using anti-UBE2D1/2/3/4 antibody (orb692216). Overlay histogram showing A431 cells stained with orb692216 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-UBE2D1/2/3/4 Antibody (orb692216, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Flow Cytometry analysis of RAW264.7 cells using anti-UBE2D1/2/3/4 antibody (orb692216). Overlay histogram showing RAW264.7 cells stained with orb692216 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-UBE2D1/2/3/4 Antibody (orb692216, 1 μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Flow Cytometry analysis of RH35 cells using anti-UBE2D1/2/3/4 antibody (orb692216). Overlay histogram showing RH35 cells stained with orb692216 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-UBE2D1/2/3/4 Antibody (orb692216, 1 μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.