SQSTM1/p62 Antibody (monoclonal, 3H11)

• Catalog Number: orb570318
• Category: Antibodies
• Description: SQSTM1/p62 Antibody (monoclonal, 3H11)
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 3H11
• Tested applications: FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG2a
• Immunogen: A synthetic peptide corresponding to a sequence at the N-terminus of human SQSTM1/p62 (69-96aa DEDGDLVAFSSDEELTMAMSYVKDDIFR), identical to the related mouse and rat sequences.
• Concentration: 0
• Dilution range: Western blot, 0.1-0.5μg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human, Mouse, Rat, By Heat Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 62 kDa
• UniProt ID: Q13501
• 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.
• Alternative names: Sequestosome-1; EBI3-associated protein of 60 kDa;
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. By Heat: Boiling the paraffin sections in 10mM citrate buffer, pH6.0, for 20mins is required for the staining of formalin/paraffin sections. 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 SQSTM1 using anti-SQSTM1 antibody (orb570318). 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: human CCRF-CEM whole cell lysates Lane 2: human HepG2 whole cell lysates Lane 3: human HT1080 whole cell lysates Lane 4: human SW620 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 mouse anti-SQSTM1 antigen affinity purified monoclonal antibody (Catalog # orb570318) 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 # orb90502) with Tanon 5200 system. A specific band was detected for SQSTM1 at approximately 62KD. The expected band size for SQSTM1 is at 62KD.

Western blot analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 brain tissue lysates Lane 2: rat liver tissue lysates Lane 3: rat PC-12 whole cell lysates Lane 4: rat RH35 whole cell lysates Lane 5: mouse brain tissue lysates Lane 6: mouse liver tissue lysates Lane 7: mouse NIH/3T3 whole cell lysates Lane 8: mouse RAW246.7 whole cell lysates Lane 9: mouse Neuro-2a 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 mouse anti-SQSTM1 antigen affinity purified monoclonal antibody (Catalog # orb570318) 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 # orb90502) with Tanon 5200 system. A specific band was detected for SQSTM1 at approximately 62KD. The expected band size for SQSTM1 is at 62KD.

IHC analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 (orb570318) 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 # orb90443) with DAB as the chromogen.

IHC analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 (orb570318) 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 # orb90443) with DAB as the chromogen.

IHC analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 (orb570318) 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 # orb90443) with DAB as the chromogen.

IHC analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 (orb570318) 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 # orb90443) with DAB as the chromogen.

IHC analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). 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 (orb570318) 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 # orb90443) with DAB as the chromogen.

IF analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). SQSTM1 was detected in immunocytochemical section of A431 cell. 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 2μg/mL mouse anti-SQSTM1 Antibody (orb570318) overnight at 4°C. DyLight®488 Conjugated Goat Anti-mouse 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 A549 cells using anti-SQSTM1 antibody (orb570318). Overlay histogram showing A549 cells stained with orb570318 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-SQSTM1 Antibody (orb570318, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Flow Cytometry analysis of PC-3 cells using anti-SQSTM1 antibody (orb570318). Overlay histogram showing PC-3 cells stained with orb570318 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-SQSTM1 Antibody (orb570318, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

IF analysis of SQSTM1 using anti-SQSTM1 antibody (orb570318). SQSTM1 was detected in immunocytochemical section of MCF7 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 2μg/mL mouse anti-SQSTM1 Antibody (orb570318) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse 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.