p62/SQSTM1 Antibody - BSA Free

Novus Biologicals | Catalog # NBP1-42822

Novus Biologicals
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Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse, Rat

Applications

Validated:

Knockout Validated, Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Simple Western

Cited:

Western Blot, Flow Cytometry, Flow Cytometry Control

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all p62/SQSTM1 Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to an internal region of the human p62/SQSTM1 protein (within residues 400-450). [Swiss-Prot Q13501]

Localization

Cytoplasm. Late endosome. Nucleus.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for p62/SQSTM1 Antibody - BSA Free

Knockout Validated: p62/SQSTM1 Antibody - BSA Free [NBP1-42822]

Western Blot: p62/SQSTM1 Antibody - BSA Free [NBP1-42822]

Western Blot: p62/SQSTM1 Antibody [NBP1-42822] - Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and p62/SQSTM1 knockout (KO) HeLa cell line. PVDF membrane was probed with Rabbit Anti-Human p62/SQSTM1 polyclonal Antibody (Catalog # NBP1-42822) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog #HAF008). Specific band was detected for p62/SQSTM1 at approximately 60 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. This experiment was conducted under reducing conditions.
Western Blot: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

Western Blot: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

Western Blot: p62/SQSTM1 Antibody [NBP1-42822] - HeLa cells were treated with (+) or without 50 uM (-) of Chloriquine (CQ) for 24 hours. Total cell lysates were prepared and separated on a 12% gel by SDS-PAGE. Protein was transferred to PVDF membrane and blocked in 5% non-fat milk. The membrane was then probed with 2 ug/ml anti-p62/SQSMT1 in 1% milk and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the upregulation of p62 (arrowhead) in response to chloroquine treatment and the blockage of autophagy.
Simple Western: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

Simple Western: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

Simple Western: p62/SQSTM1 Antibody [NBP1-42822] - Simple Western lane view shows a specific band for p62/SQSTM1 in 0.5 mg/ml of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Western Blot: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

Western Blot: p62/SQSTM1 AntibodyBSA Free [NBP1-42822]

p62-SQSTM1-Antibody-Western-Blot-NBP1-42822-img0010.jpg

Applications for p62/SQSTM1 Antibody - BSA Free

Application
Recommended Usage

Flow Cytometry

reported in scientific literature (PMID 35451674)

Immunocytochemistry/ Immunofluorescence

1:50

Simple Western

1:25

Western Blot

1:2000
Application Notes
In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in HeLa lysate 0.5 mg/mL and HeLa lysate 0.1 mg/mL; separated by Size; antibody dilution of 1:200, 1:25; apparent MW was 110 kDa.

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS and 30% Glycerol

Format

BSA Free

Preservative

0.1% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: p62/SQSTM1

p62/SQSTM1 (ubiquitin-binding protein p62/Sequestrosome-1) is an intracellular protein (theoretical molecular weight 47.7 kDa) which localizes to the cytoplasm, nucleus, autophagosomes and lysosomes in all tissues (1). Structurally, p62/SQSTM1 consists of multiple domains (e.g., Phox1 and Bem1p-PB1, zinc finger-ZZ, TRAF6 binding domain-TB, Keap1-interacting region-KIR, LC3 interacting region-LIR and ubiquitin-associated domain-UBA) for interaction with various protein targets. Additional domains include nuclear export (NES) and nuclear localization signals (NLS1 and NLS2). p62/SQSTM1 is a multifunctional scaffold protein which forms oligomers with itself or with other proteins through interactions with PB1 domains.

Abnormal function of p62/SQSTM1 is associated with a range of disease states such as neurodegeneration, cancer, and metabolic disorders (2). Mutations in the p62/SQSTM1 sequence have been linked to Paget's disease of the bone, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration. In Parkinson's disease, p62/SQSTM1 has been linked to microglia activation and subsequent neuroinflammation (3). Functionally, p62/SQSTM1 is involved in a broad range of cellular processes such as amino acid sensing by interaction with mTORC1, oxidative stress response through interaction with Keap1, and targeting cargo for autophagy by interacting with ubiquitin labeled proteins (1).

To induce selective autophagy, p62/SQSTM1 forms long oligomers or helical filaments which interact with LC3 and ubiquitin labeled proteins and lead to the initiation of the autophagosome formation (2). p62/SQSTM1 is not only a selective autophagy receptor but also an autophagy substrate, as its engulfed by the autophagosome and degraded by the autophagolysosome. Monitoring LC3 levels is the standard for assessing autophagic flux, however monitoring p62/SQSTM1 levels by Western blot in the presence and absence of autophagy inhibitors (e.g., Chloroquine) is also a common practice (4). Besides its activity as a selective autophagy receptor, p62/SQSTM1 also plays a role as an adaptor in signaling cascades leading to NFkB activation downstream of TNF-R, IL-1 beta R, TrkA and p75NTR. Briefly, for NFkB signaling downstream of the TNF-R activation, p62/SQSTM1 engages RIP1 kinase and PKC iota/lambda through the ZZ and PB1 domains, respectively (5).

References

1.Katsuragi, Y., Ichimura, Y., & Komatsu, M. (2015). P62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS Journal. https://doi.org/10.1111/febs.13540

2. Sanchez-Martin, P., & Komatsu, M. (2018). p62/SQSTM1 - Steering the cell through health and disease. Journal of Cell Science. https://doi.org/10.1242/jcs.222836

3. Yao, L., Zhu, Z., Wu, J., Zhang, Y., Zhang, H., Sun, X.,... Lu, G. (2019). MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease. The FASEB Journal. https://doi.org/10.1096/fj.201900363r

4 Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Arozena, A. A.,... Zughaier, S. M. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. https://doi.org/10.1080/15548627.2015.1100356

5. Bitto, A., Lerner, C. A., Nacarelli, T., Crowe, E., Torres, C., & Sell, C. (2014). p62/SQSTM1 at the interface of aging, autophagy, and disease. Age. https://doi.org/10.1007/s11357-014-9626-3

Long Name

Sequestosome 1

Alternate Names

A170, EBIAP, ORCA, OSIL, PDB3, Sequestosome 1, SQSTM1, ZIP3

Entrez Gene IDs

8878 (Human)

Gene Symbol

SQSTM1

UniProt

Q13501

Additional p62/SQSTM1 Products

Product Documents for p62/SQSTM1 Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for p62/SQSTM1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Related Research Areas

Autophagy
NFkB Pathway

Citations for p62/SQSTM1 Antibody - BSA Free

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Protocols

View specific protocols for p62/SQSTM1 Antibody - BSA Free (NBP1-42822):

p62/SQSTM1 Antibody:
Procedure Guide for NBP1-42822 - SQSTM1 Antibody
Western Blot Protocol
1. Perform SDS-PAGE (4-12% MOPS) on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer
apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of
proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations
and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% BSA in TBS + Tween, 1 hour at RT.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10
minutes each.
7. Dilute the rabbit anti-SQSTM1 primary antibody (NBP1-42822) in blocking buffer and incubate 1 hour at room
temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10
minutes each.
9. Apply the diluted rabbit-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers
instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as
required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions (Pierce ECL).
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided
it does not interfere with antibody-antigen binding.

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