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    products/primary-antibodies/sqstm1--p62-antibody-ab91526.pdf

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Anti-SQSTM1 / p62 antibody (ab91526)

  • Datasheet
  • SDS
Reviews (6)Q&A (5)References (247)

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Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)

Key features and details

  • Rabbit polyclonal to SQSTM1 / p62
  • Suitable for: ICC/IF, WB, IHC-P
  • Reacts with: Mouse, Rat, Human
  • Isotype: IgG

Get better batch-to-batch reproducibility with a recombinant antibody

Product image
Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
  • Research with confidence – consistent and reproducible results with every batch
  • Long-term and scalable supply – powered by recombinant technology for fast production
  • Success from the first experiment – confirmed specificity through extensive validation
  • Ethical standards compliant – production is animal-free

Overview

  • Product name

    Anti-SQSTM1 / p62 antibody
    See all SQSTM1 / p62 primary antibodies
  • Description

    Rabbit polyclonal to SQSTM1 / p62
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human SQSTM1/ p62 (C terminal).
    Database link: Q13501

  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at +4°C.
  • Storage buffer

    pH: 7.2
    Preservative: 0.02% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFkB Pathway
    • Signal Transduction
    • Protein Trafficking
    • Vesicle Transport
    • Regulation
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Polymerase associated factors
    • Pol II Transcription
    • Other
    • Cardiovascular
    • Heart
    • Autophagy
    • Autophagosome
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Autophagy and mitophagy
    • Autophagosome
    • Cancer
    • Cell Death
    • Autophagy
    • Autophagosome
    • Neuroscience
    • Processes

Associated products

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

    • Recombinant Human SQSTM1 / p62 protein (ab95320)
  • Related Products

    • Prestained Protein Ladder – Broad molecular weight (10-245 kDa) (ab116028)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab91526 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF (1)
Use at an assay dependent concentration.
WB (4)
Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 47 kDa).

Only mouse and human species are validated in WB. Rat is not a validated species.

IHC-P
Use a concentration of 2 - 5 µg/ml.
Notes
ICC/IF
Use at an assay dependent concentration.
WB
Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 47 kDa).

Only mouse and human species are validated in WB. Rat is not a validated species.

IHC-P
Use a concentration of 2 - 5 µg/ml.

Target

  • Function

    Adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. May play a role in titin/TTN downstream signaling in muscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates the interaction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels.
  • Tissue specificity

    Ubiquitously expressed.
  • Involvement in disease

    Defects in SQSTM1 are a cause of Paget disease of bone (PDB) [MIM:602080]. PDB is a metabolic bone disease affecting the axial skeleton and characterized by focal areas of increased and disorganized bone turn-over due to activated osteoclasts. Manifestations of the disease include bone pain, deformity, pathological fractures, deafness, neurological complications and increased risk of osteosarcoma. PDB is a chronic disease affecting 2 to 3% of the population above the age of 40 years.
  • Sequence similarities

    Contains 1 OPR domain.
    Contains 1 UBA domain.
    Contains 1 ZZ-type zinc finger.
  • Domain

    The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinated substrates. Mediates the interaction with TRIM55.
    The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5 and NBR1.
    The ZZ-type zinc finger mediates the interaction with RIPK1.
  • Post-translational
    modifications

    Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN.
  • Cellular localization

    Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes to the sarcomeric band (By similarity). Localizes to late endosomes. May also localize to the nucleus. Accumulates in neurofibrillary tangles and in Lewy bodies of neurons from individuals with Alzheimer and Parkinson disease respectively. Enriched in Rosenthal fibers of pilocytic astrocytoma. In liver cells, accumulates in Mallory bodies associated with alcoholic hepatitis, Wilson disease, indian childhood cirrhosis and in hyaline bodies associated with hepatocellular carcinoma.
  • Target information above from: UniProt accession Q13501 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 8878 Human
    • Entrez Gene: 18412 Mouse
    • Entrez Gene: 113894 Rat
    • Omim: 601530 Human
    • SwissProt: Q13501 Human
    • SwissProt: Q64337 Mouse
    • SwissProt: O08623 Rat
    • Unigene: 709030 Human
    • Unigene: 40828 Mouse
    • Unigene: 107103 Rat
    see all
  • Alternative names

    • A170 antibody
    • DMRV antibody
    • EBI 3 associated protein of 60 kDa antibody
    • EBI 3 associated protein p60 antibody
    • EBI3 associated protein of 60 kDa antibody
    • EBI3 associated protein p60 antibody
    • EBI3-associated protein of 60 kDa antibody
    • EBIAP antibody
    • FTDALS3 antibody
    • MGC127197 antibody
    • ORCA antibody
    • OSF-6 antibody
    • Osi antibody
    • OSIL antibody
    • Oxidative stress induced like antibody
    • p60 antibody
    • p62 antibody
    • p62B antibody
    • Paget disease of bone 3 antibody
    • PDB 3 antibody
    • PDB3 antibody
    • Phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa antibody
    • Phosphotyrosine independent ligand for the Lck SH2 domain p62 antibody
    • Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa antibody
    • PKC-zeta-interacting protein antibody
    • Protein kinase C-zeta-interacting protein antibody
    • Sequestosome 1 antibody
    • Sequestosome-1 antibody
    • SQSTM 1 antibody
    • SQSTM_HUMAN antibody
    • Sqstm1 antibody
    • STAP antibody
    • STONE14 antibody
    • Ubiquitin binding protein p62 antibody
    • Ubiquitin-binding protein p62 antibody
    • ZIP 3 antibody
    • ZIP antibody
    • ZIP3 antibody
    see all

Images

  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    All lanes : Anti-SQSTM1 / p62 antibody (ab91526) at 0.5 µg/ml

    Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate
    Lane 2 : A431 (human epidermoid carcinoma cell line) cell lysate
    Lane 3 : A549 (human lung carcinoma cell line) cell lysate
    Lane 4 : CaCo-2 (human colorectal adenocarcinoma cell line) cell lysate
    Lane 5 : Daudi (human Burkitt's lymphoma cell line) cell lysate
    Lane 6 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 7 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
    Lane 8 : K562 (human chronic myelogenous leukemia cell line from bone marrow) cell lysate
    Lane 9 : MCF7 (human breast adenocarcinoma cell line) cell lysate
    Lane 10 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate
    Lane 11 : SK-N-SH (human neuroblastoma cell line) cell lysate
    Lane 12 : THP-1 (human monocytic leukemia cell line) cell lysate
    Lane 13 : NIH/3T3 (mouse embryo fibroblast cell line) cell lysate
    Lane 14 : L1210 (mouse lymphocytic leukemia cell line) cell lysate

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG (HRP) at 1/10000 dilution

    Predicted band size: 47 kDa



    Diluting buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)Han, Z. et al PLoS One. 2014 Jan 23;9(1):e86838. doi: 10.1371/journal.pone.0086838. eCollection 2014 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse heart tissue labelling SQSTM1 / p62 with ab91526. Heat mediated antigen retrieval was performed. The tissue sections were then blocked with 10% goat serum in PBS, and incubated with primary antibody overnight at 4°C. Sections were incubated with secondary antibody for 1 h at room temperature, incubated with avidin-biotin complex for 1 h at room temperature, rinsed with PBS and then treated with 0.5 mg/mL DAPI.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)

    Immunofluorescent analysis of 4% paraformaldehyde fixed Mouse Spleen Tissue labeling SQSTM1 / p62 with ab91526 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue).

  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    All lanes : Anti-SQSTM1 / p62 antibody (ab91526) at 0.5 µg/ml

    Lane 1 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate - untreated
    Lane 2 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate treated with 0.3 µg/mL LPS for 3 hours
    Lane 3 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate treated with 0.3 µg/mL LPS for 6 hours
    Lane 4 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate treated with 0.3 µg/mL LPS for 24 hours

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG (HRP) at 1/10000 dilution

    Predicted band size: 47 kDa



    Diluting buffer: 5% NFDM/TBST.

    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)

    A431 (human epidermoid carcinoma cell line) cells stained for SQSTM1 / p62 (green) using ab91526 at 20 µg/ml in ICC/IF.

  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)

    Immunofluorescence of SQSTM1 / p62 in Rat Spleen cells using ab91526 at 20 ug/ml.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)

    Paraffin-embedded human spleen tissue stained for SQSTM1/p62 using ab91526 at 5 µg/ml in immunohistochemical analysis.

  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Lane 1 : Anti-SQSTM1 / p62 antibody (ab91526) at 1 µg/ml
    Lane 2 : Anti-SQSTM1 / p62 antibody (ab91526) at 2 µg/ml

    All lanes : Human spleen tissue lysate

    Lysates/proteins at 15 µg per lane.

    Predicted band size: 47 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody (ab91526)Image from Traver MK et al, J Biol Chem. 2011 Sep 2;286(35):30471-80. Epub 2011 Jul 12, Fig 5. DOI 10.1074/jbc.M111.251967 September 2, 2011 The Journal of Biological Chemistry, 286, 30471-30480.
    ab91526 at a 1/500 dilution staining SQSTM1/ p62 in wild type murine embryonic fibroblasts by Immunocytochemistry/ Immunofluorescence.
  • Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Western blot - Anti-SQSTM1 / p62 antibody (ab91526)
    Western blot analysis of SQSTM1 expression in K562 cell lysate with ab91526 at 1µg/ml in (A) the absence and (B) the presence of blocking peptide.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)

    Rat spleen tissue stained for SQSTM1 / p62 using ab91526 at 5 μg/ml in immunohistochemical analysis.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody (ab91526)

    Mouse spleen tissue stained for SQSTM1 / p62 using ab91526 at 2 μg/ml in immunohistochemical analysis.

Protocols

  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (247)

Publishing research using ab91526? Please let us know so that we can cite the reference in this datasheet.

ab91526 has been referenced in 247 publications.

  • Marino-Merlo F  et al. Caspase-8 is required for HSV-1-induced apoptosis and promotes effective viral particle release via autophagy inhibition. Cell Death Differ 30:885-896 (2023). PubMed: 36418547
  • Geismann C  et al. NF-κB/RelA controlled A20 limits TRAIL-induced apoptosis in pancreatic cancer. Cell Death Dis 14:3 (2023). PubMed: 36596765
  • Filippone A  et al. Sodium Propionate Contributes to Tumor Cell Growth Inhibition through PPAR-γ Signaling. Cancers (Basel) 15:N/A (2022). PubMed: 36612214
  • Cui Y  et al. Electroacupuncture attenuates spared nerve injury-induced neuropathic pain possibly by promoting the progression of AMPK/mTOR-mediated autophagy in spinal microglia. Ann Transl Med 10:1278 (2022). PubMed: 36618785
  • Wang M  et al. Neuroprotective Mechanism of Icariin on Hypoxic Ischemic Brain Damage in Neonatal Mice. Oxid Med Cell Longev 2022:1330928 (2022). PubMed: 36425058
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 11 Abreviews or Q&A

Immunohistochemistry (Frozen sections) abreview for Anti-SQSTM1 / p62 antibody

Average
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (skeletal muscle biopsy)
Permeabilization
No
Specification
skeletal muscle biopsy
Blocking step
PNB (PerkinElmer) as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 22°C
Fixative
Acetone
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Mar 15 2023

Immunocytochemistry/ Immunofluorescence abreview for Anti-SQSTM1 / p62 antibody

Excellent
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
African green monkey Cell (Kidney)
Permeabilization
Yes - 0.2% Triron X100
Specification
Kidney
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Methanol
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Sep 27 2017

Western blot abreview for Anti-SQSTM1 / p62 antibody

Good
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Gastrocnemius)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
40 µg
Specification
Gastrocnemius
Blocking step
Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Apr 20 2015

Western blot abreview for Anti-SQSTM1 / p62 antibody

Good
Abreviews
Abreviews
abreview image
Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Cell lysate - whole cell (COV434 cell line)
Specification
COV434 cell line
Blocking step
Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Feb 13 2015

Western blot abreview for Anti-SQSTM1 / p62 antibody

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Reduced Denaturing (4-20%)
Sample
Mouse Tissue lysate - whole (Lung)
Specification
Lung
Treatment
Bafilomycin
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Alan Chang

Verified customer

Submitted Mar 20 2014

Question

High background with ab91526 and mouse aorta samples.

Read More

Abcam community

Verified customer

Asked on Sep 07 2012

Answer

Thank you very much for your call today and for letting us know about the trouble with ab91526.

As we discussed, please keep me updated about the results after reducing the protein loading to 20, 40, and 60 ug per lane. If the results don't improve I'll be happy to send a replacement or issue a credit or refund.

I look forward to hearing from you. If you have any questions or need anything else, let me know and I'll be happy to help. Have a nice day!

Read More

Abcam Scientific Support

Answered on Sep 07 2012

Question

Inquiry: The customer stored ab91526 xx. Three weeks ago the customer used about 20microliters of the antibody and left the rest of the ab at 4C. Yesterday the customer opened the vial and found that the solution of the ab evaporated. She saw white pellet on the bottom of the vial. Please advise. Thank you. Kind Regards,

Read More

Abcam community

Verified customer

Asked on Jun 26 2012

Answer

Thank you for your inquiry.
If the cap of the vial has not been screwed tightly back again, it is very well possible that the remaining 80ul have been evaporated during the 3 weeks. Please recommend the customer to tightly screw the cap on the vials when storing them. In cases of doubt, the customer can also use parafilm to avoid any evaporation. This is however not necessary in general.

Read More

Abcam Scientific Support

Answered on Jun 26 2012

Question

thank you for your prompt answer. Here below the answers to your questions. I hope this could help in solving the problem.

1. Do you know the lot number and order number of each of the antibodies?
The lot numbers are the following: Apg7 Lot GR54674-4

p62 GR7582-8

While for the order number at the moment I can’t provide you any information as the technician who ordered the antibodies for me is now on holiays.

2. How was the sample collected and treated prior to loading on the gel? What lysis buffer was used? Were any protease inhibitors used? Was the sample boiled prior to loading? Was any reducing agent used?
Samples were first stored at -80 (after dissection of the animal), thawed in ice. Lysis buffer added and samply lysed by sonication with two pulses for 5 sec.
Lysis buffer: SDS 1% (or triton X 100 1% in a second experiment), EDTA 1mM, NaCl 50mM and TrisHcl pH 7.4 100mM.
Complete protease and phospatase inhibitors were freshly added to the lysate.
Samples were boiled for 10min at 95C after adding loading buffer.
No reducing agents were used.

3. How was the blocking performed? Time/temperature? Was any detergent included in the blocking buffer?
Blocking was done in different blocking buffers: milk 5% or BSA 5% or NGS 5%. 1h at room temperature. Blocking buffers were diluted in TBS + 0.1% Tween 20.

4. How was the primary antibody incubated with the membrane (time/temperature)? What diluent was used with each of the antibodies?
Antibodies were incubated O/N at 4C. Antibodies were diluted in milk 2%, BSA 5% or NGS 5% in TBS + 0.1% Tween 20.

5. What washing steps were employed and with what buffer?
Washing steps: 3x 10 min in TBS + 0.1% Tween 20.

6. Were the membranes freshly probed or was the same membrane used and stripped between each antibody? If so, how was this procedure performed?
Membranes were all freshly probed.

7. Secondary antibody:goat anti rabbit, used 1:10’000 in milk 2%, BSA 5% or NGS 5% in TBS + 0.1% Tween 20. 1h at room temperature.

All the best,

Read More

Abcam community

Verified customer

Asked on Mar 28 2012

Answer

Thank you for providing that information.

Having reviewed the protocol used, I believe there are afew things that maybe worthtrying in order toimprove the specificity observed.I would suggest using a reducing agent in the loading buffer to reduce the aggregation which may be occurring (especially with ab91526). Either DTT or b-mecaptoethanol. It would also be worth reducing the temperature at which the protein is denatured. Certain proteins are prone to aggregation, which can be reduced by heating to 70 degrees for 10 minutes instead of 95. I would also suggest reducing the concentration used slightly (ab53255 down to 1/500 and ab91526 to 1/1000) to see if this improves the binding as well as reducing the amount of protein loaded (down to 40 ug/lane).

Has a "no primary" control been performed? This may be worthwhile, just to rule out any non-specificity relating to the secondary antibody used.

Should these suggestions not improve the results please do let me know and I'll look into if we have any alternative lots of these antibodiesor different antibodies altogetherwhich you may be interested in trying.

Read More

Abcam Scientific Support

Answered on Mar 28 2012

Question

Dear Madam/Sir,

I recently purchased two products from your primary antibody catalogue and both of them gave me problems in the detection of the expected protein.

Here following the explanation of my technical issues and in attachment the corresponding Western Blots, done by loading 100ug of mouse brain lysate (hypothalamic region):

Antibody against Apg7 (a b53255):The expected band should appear at 78kDa. I can detect a band at that height only using BSA or NGS as blocking agents. However other unspecific bands are detected as well. (see attachment)

Antibody against p62 (a b91526):Similar situation as above. The protein should be expected at 47kDa, but several other bands are present and signal at the right height appears only in BSA and NGS blocking conditions. However, in this case the signal given from unspecific bands is even stronger. (see attachment)

In both cases the images available in your datasheet show specific signal at the expected height and a clear western blot.

Seen these discrepancies, although using the same experimental conditions,I would appreciate a technical help to solve this problem or some suitable alternative.

Unfortunately the presence of so many unspecific bands would not allow me to produce consistent and robust scientific data, thus influencing the output of my research.

I thank you in advance for your help.

Best regards

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Abcam community

Verified customer

Asked on Mar 26 2012

Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewedthe information you have provided, I would like to confirm a few further details in order to understand the procedure used more fully and hopefully provide more specific protocol tips:

1. Do you know the lot number and order number of each of the antibodies?

2. How was the sample collected and treated prior to loading on the gel? What lysis buffer was used? Were any protease inhibitors used? Was the sample boiled prior to loading? Was any reducing agent used?

3. How was the blocking performed? Time/temperature? Was any detergent included in the blocking buffer?

4. How was the primary antibody incubated with the membrane (time/temperature)? What diluent was used with each of the antibodies?

5. What washing steps were employed and with what buffer?

6. Were the membranes freshly probed or was the same membrane used and stripped between each antibody? If so, how was this procedure performed?

Hopefully with this information I will be ableto suggest modificationswhich will improve the specificity currently observed.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I look forward to receiving your reply.

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Abcam Scientific Support

Answered on Mar 26 2012

Question

It is indicated in the datasheet of ab91526  that predicted molecular weight is 47 kDa. In the  image of the abreview specific band of 62 kDa is observed.   Please clarify this discrepancy.   Thanks in advance for your assistance and reply.

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Abcam community

Verified customer

Asked on Feb 27 2012

Answer

Thank you for contacting us.

There are number of reasons which may affect the apparent band size from one western blot to the next. In this particular case those may include the fact that this protein undergoes a number of post translational modifications which may greatly affect its size. The Abreview used a different species than the datasheet. The image in the Abreview was created by a customer and as such we do not have the exact protocol that they used including but not limited to gels and protein markers used and sample treatment.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam Scientific Support

Answered on Feb 27 2012

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