Figure 1. Western blot analysis of RNF111 using anti-RNF111 antibody (MBS1758636).
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: mouse thymus 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-RNF111 antigen affinity purified polyclonal antibody (#MBS1758636) at 0.5ug/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 (#MBS176460) with Tanon 5200 system. A specific band was detected for RNF111 at approximately 109 kDa. The expected band size for RNF111 is at 109 kDa.
Figure 2. IHC analysis of RNF111 using anti-RNF111 antibody (MBS1758636).
RNF111 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2ug/ml rabbit anti-RNF111 Antibody (MBS1758636) overnight at 4 degree C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (#MBS176453) with DAB as the chromogen.
Figure 3. IHC analysis of RNF111 using anti-RNF111 antibody (MBS1758636).
RNF111 was detected in a paraffin-embedded section of rat spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2ug/ml rabbit anti-RNF111 Antibody (MBS1758636) overnight at 4 degree C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (#MBS176453) with DAB as the chromogen.
Figure 4. IF analysis of RNF111 using anti-RNF111 antibody (MBS1758636) and anti-Beta Tubulin antibody (M01857-3).
RNF111 was detected in immunocytochemical section of A549 cell. 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 5ug/mL rabbit anti-RNF111 Antibody (MBS1758636) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4 degree C. DyLight488 Conjugated Goat Anti-Rabbit IgG (MBS177405) and DyLight550 Conjugated Goat Anti-Mouse IgG (BA1133) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37 degree C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 5. Flow Cytometry analysis of SH-SY5Y cells using anti-RNF111 antibody (MBS1758636).
Overlay histogram showing SH-SY5Y cells stained with MBS1758636 (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-RNF111 Antibody (MBS1758636, 1ug/1x106 cells) for 30 min at 20 degree C. DyLight488 conjugated goat anti-rabbit IgG (MBS177405, 5-10ug/1x106 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1ug/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
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