Anti-SQSTM1/p62 Antibody Picoband™ (monoclonal, 3H11)

Figure 1. Western blot analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
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 HepG2 whole cell lysates,
Lane 2: human A549 whole cell lysates,
Lane 3: human RT4 whole cell lysates,
Lane 4: human Hela whole cell lysates,
Lane 5: human SiHa whole cell lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: rat RH35 whole cell lysates,
Lane 8: mouse NIH/3T3 whole cell lysates,
Lane 9: mouse RAW264.7 whole cell 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 mouse anti-SQSTM1 antigen affinity purified monoclonal antibody (Catalog # M00300-1) 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-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for SQSTM1 at approximately 62 kDa. The expected band size for SQSTM1 is at 48 kDa.

Figure 2. IHC analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in section of rat intestine tissues. 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 μg/ml mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. Biotinylated goat anti-mouse 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 # SA1021) with DAB as the chromogen.

Figure 3. IHC analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in section of human colon cancer tissues. 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 μg/ml mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. Biotinylated goat anti-mouse 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 # SA1021) with DAB as the chromogen.

Figure 4. IHC analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in section of human mammary cancer tissues. 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 μg/ml mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. Biotinylated goat anti-mouse 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 # SA1021) with DAB as the chromogen.

Figure 5. IHC analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in section of human lung cancer tissues. 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 μg/ml mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. Biotinylated goat anti-mouse 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 # SA1021) with DAB as the chromogen.

Figure 6. IHC analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in section of mouse intestine tissues. 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 μg/ml mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. Biotinylated goat anti-mouse 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 # SA1021) with DAB as the chromogen.

Figure 7. IF analysis of SQSTM1 using anti- SQSTM1 antibody (M00300-1).
SQSTM1 was detected in immunocytochemical section of MCF7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL mouse anti- SQSTM1 Antibody (M00300-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) 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.

Figure 8. IF analysis of SQSTM1 using anti-SQSTM1 antibody (M00300-1).
SQSTM1 was detected in immunocytochemical section of A431 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL mouse anti-SQSTM1 Antibody (M00300-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-mouse IgG (BA1126) 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.

Figure 9. Flow Cytometry analysis of A549 cells using anti-SQSTM1 antibody (M00300-1).
Overlay histogram showing A549 cells stained with M00300-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-SQSTM1 Antibody (M00300-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 10. Flow Cytometry analysis of PC-3 cells using anti-SQSTM1 antibody (M00300-1).
Overlay histogram showing PC-3 cells stained with M00300-1 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-SQSTM1 Antibody (M00300-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.