Figure 1. Western blot analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
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 30ug of sample under reducing conditions.
Lane 1: human placenta tissue lysates
Lane 2: human A549 whole cell lysates
Lane 3: human Hela whole cell lysates
Lane 4: human CCRF-CEM whole cell lysates
Lane 5: human U-87MG whole cell lysates
Lane 6: human SY-SY5Y whole cell lysates
Lane 7: monkey heart tissue 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-EHD3 antigen affinity purified polyclonal antibody (Catalog # MBS1753665) at 0. 25 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 EHD3 at approximately 70KD. The expected band size for EHD3 is at 70KD.
Figure 2. Western blot analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
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 30ug of sample under reducing conditions.
Lane 1: rat heart tissue lysates
Lane 2: rat brain tissue lysates
Lane 3: rat kidney tissue lysates
Lane 4: rat liver tissue lysates
Lane 5: mouse heart tissue lysates
Lane 6: mouse brain tissue lysates
Lane 7: mouse kidney tissue lysates
Lane 8: mouse liver tissue 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-EHD3 antigen affinity purified polyclonal antibody (Catalog # MBS1753665) at 0. 25 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 EHD3 at approximately 70KD. The expected band size for EHD3 is at 70KD.
Figure 3. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human ovarian serous adenocarcinoma 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-EHD3 Antibody (MBS1753665) 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 EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human ovarian serous adenocarcinoma 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-EHD3 Antibody (MBS1753665) 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 EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human renal clear cell carcinoma 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-EHD3 Antibody (MBS1753665) 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. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human breast 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-EHD3 Antibody (MBS1753665) 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 7. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human lung 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-EHD3 Antibody (MBS1753665) 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 8. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human rectal 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-EHD3 Antibody (MBS1753665) 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 9. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of human gastric adenocarcinoma 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-EHD3 Antibody (MBS1753665) 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 10. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of mouse brain 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-EHD3 Antibody (MBS1753665) 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 11. IHC analysis of EHD3 using anti-EHD3 antibody (MBS1753665).
EHD3 was detected in paraffin-embedded section of rat brain 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-EHD3 Antibody (MBS1753665) 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 12. IF analysis of EHD3 using anti- EHD3 antibody (MBS1753665).
EHD3 was detected in immunocytochemical section of MCF-7 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- EHD3 Antibody (MBS1753665) 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 13. Flow Cytometry analysis of MCF-7 cells using anti-EHD3 antibody (MBS1753665).
Overlay histogram showing MCF-7 cells stained with MBS1753665 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-EHD3 Antibody (MBS1753665, 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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