Mediator of RNA polymerase II transcription subunit 8
Application Dilute:
Western blot, 0.1-0.5µg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1µg/ml, Mouse, Rat Immunocytochemistry/Immunofluorescence, 2µg/ml, Human Flow Cytometry (Fixed), 1-3µg/1x10 6 cells, Human ELISA, 0.1-0.5µg/ml
Flow Cytometry analysis of 293T cells using anti-MED8 antibody. Overlay histogram showing 293T cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-MED8 Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
IF analysis of MED8 and Tubulin beta usi
IF analysis of MED8 and Tubulin beta using anti-MED8 antibody and anti-Tubulin beta antibody. MED8 and Tubulin beta were detected in immunocytochemical section of U87 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL rabbit anti-MED8 antibody and mouse anti-Tubulin beta Antibody overnight at 4C. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight488 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IHC analysis of MED8 using anti-MED8 antibody. MED8 was detected in paraffin-embedded section of mouse testis tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 ug/ml rabbit anti-MED8 Antibody overnight at 4 Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
IHC analysis of MED8 using anti-MED8 antibody. MED8 was detected in paraffin-embedded section of rat testis tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 ug/ml rabbit anti-MED8 Antibody overnight at 4 Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Western blot analysis of MED8 using anti-MED8 antibody. 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 THP-1 whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human U251 whole cell lysates, Lane 4: human Caco-2 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: rat liver tissue lysates, Lane 7: mouse brain tissue lysates, Lane 8: mouse liver 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-MED8 antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4C, 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 MED8 at approximately 29 kDa. The expected band size for MED8 is at 29 kDa.
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