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<strong>Figure 1 KO Validation in HEK293T Cells</strong><br> Loading: 10 μg of HEK293T WT cell lysates or SQSTM1 KO cell lysates. Antibodies: SQSTM1 5449 (1 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2. Immunofluorescence Validation of SQSTM1 In HeLa Cells </strong><br> Immunofluorescent analysis of PFA-fixed HeLa cells labeling SQSTM1 with 5449 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/1000 dilution (red) and Hoechst staining (blue). Alpha tubulin was stained with anti-alpha tubulin antibody following by goat anti-mouse IgG secondary antibody (green). Images were captured with confocal microscopy.
<strong>Figure 3 Western Blot Validation in Cell Lines</strong><br>Loading: 15 µg of lysates per lane.Antibodies: SQSTM1 5449 (0.5 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Induced Expression Validation in Mouse Macrophage Cells</strong><br>Loading: 15 µg of lysates per lane.Antibodies: SQSTM1 5449 (0.5 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.Raw 264.7 cells were treated with LPS (0.3 µg /mL) for different time period (0-24 hrs).There was an increase in SQSTM1 protein expression overtime after LPS treatment.
<strong>Figure 5 Western Blot Validation in Mouse Tissues</strong><br>Loading: 15 µg of lysates per lane.Antibodies: SQSTM1 5449 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 6 Western Blot Validation in Rat Tissues</strong><br>Loading: 15 µg of lysates per lane.Antibodies: SQSTM1 5449 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 7 Immunohistochemistry Validation of SQSTM1 in Human Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-emb edded Human Spleen Tissue using anti-SQSTM1 antibody (5449) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 8 Immunofluorescence Validation of SQSTM1 in Mouse Spleen Tissue</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed Mouse Spleen Tissue labeling SQSTM1 with 5449 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).
<strong>Figure 9 Immunohistochemistry Validation of SQSTM1 in Mouse Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Mouse Spleen Tissue using anti-SQSTM1 antibody (5449) at 2 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 10 Immunofluorescence Validation of SQSTM1 in Rat Spleen Cells</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed Rat Spleen Cells labeling SQSTM1 with 5449 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
Figure 1 KO Validation in HEK293T Cells
Loading: 10 μg of HEK293T WT cell lysates or SQSTM1 KO cell lysates. Antibodies: SQSTM1 5449 (1 μg/mL) and beta-actin 3779 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
Cell Treatment
IAV
KO
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SQSTM1 Antibody

CATALOG NUMBER: 5449

Clonality:
Polyclonal
Tested Applications:
ELISA, IHC-P, WB, ICC/IF, IHC-P/IF
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Immunogen:
SQSTM1 antibody was raised against a 13 amino acid synthetic peptide from near the carboxy terminus of human SQSTM1.
The immunogen is located within the last 50 amino acids of SQSTM1.
Conjugate:
Unconjugated
Tested Applications:
ELISA, IHC-P, WB, ICC/IF, IHC-P/IF
Application Note:
WB: 0.5-2 μg/mL; ICC/IF: 20 μg/mL; IHC: 2-5 μg/mL;.

Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry in human, mouse and rat samples; Immunocytochemistry/Immunofluorescence in human samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1407 – Mouse Skeletal Muscle Tissue
Positive Control 2:
Cat. No. 1414 – Mouse Pancreas Tissue Lysate
Positive Control 3:
Cat. No. 1464 – Rat Liver Tissue Lysate
Predicted Molecular Weight:
Predicted: 48kD
Observed: 65 kD
Validation:

KO Validation (Figure 1): Anti-SQSTM1 antibodies (5449) specificity was further verified by SQSTM1 specific knockout. SQSTM1 signal was not detected in SQSTM1 knockout HEK293T cells as compared to that in control wild type cells.

Induced Expression Validation (Figure 2): Raw 264.7 cells were treated with LPS (0.3 µg /mL) for different time period (0-24 hrs). SQSTM1 expression detected by anit-SQSTM1 antibodies (5449) was induced by LPS treatment and the expression levels were increased gradually over the period of 24 hrs.

Isoforms:
Human SQSTM1 has two isoforms, including isoform 1 (440aa, 47.7kD) and isoform 2 (356aa, 38.6kD). Mouse SQSTM1 also has two isoforms, including isoform 1 (442aa, 48.2kD) and isoform 2 (404aa, 44.2kD). Rat SQSTM1 has three isoforms, including isoform 1 (439aa, 47.7kD), isoform 2 (412aa, 45kD) and isoform 3 (234aa, 26.1kD). 5449 can detect the isoforms of human mouse and rat except the shortest rat isoform 3.
Purification:
SQSTM1 Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
SQSTM1 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
SQSTM1 antibody can be stored at 4°C up to one year. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
SQSTM1
Additional Names:
SQSTM1 Antibody: p60, p62, A170, OSIL, PDB3, ZIP3, p62B, ORCA, Sequestosome-1, EBI3-associated protein of 60 kDa, EBIAP
Protein Accession Number:
Q13501
Protein Gi Number:
74735628
Ncbi Gene Id Number:
8878
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
SQSTM1 Antibody: SQSTM1/p62 is an adapter protein which binds ubiquitin and regulates signaling cascades through ubiquitination. It may regulate the activation of NF-κB by TNF-α, nerve growth factor (NGF) and interleukin-1. SQSTM1/p62, a co-interacting protein of the atypical PKC isoforms, has a UBA domain at its C-terminal end, which binds non-covalently to polyubiquitin chain. SQSTM1's UBA domain is necessary for recruitment of polyubiquitin and aggresome formation. SQSTM1 may play a role in titin/TTN downstream signaling in muscle cells and may be involved in cell differentiation, apoptosis, immune response and regulation of K+ channels. Mutations in the ubiquitin-associated (UBA) domain of SQSTM1 commonly cause Paget's disease of bone since the UBA is necessary for aggregate sequestration and cell survival.
Background Reference 1:
Seibenhener et al. Mol. Cell. Biol.2004; 24:8055-68.
Background Reference 2:
Hocking et al. Hum. Mol. Genet.2002; 11:2735-9.
Background Reference 3:
Rousiere et al. Best Pract. Res. Clin. Rheumatol.2003; 17:1019-41.
Background Reference 4:
Layfield et al. Biochem. Soc. Trans.2004; 32:728-30.

FOR RESEARCH USE ONLY

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Disclaimer:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

CATALOG NUMBER:

5449

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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