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  1. Link

    sqstm1--p62-antibody-2c11-bsa-and-azide-free-ab56416.pdf

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Validated using a knockout cell line

Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

  • Datasheet
Reviews (36)Q&A (4)References (731)

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Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Immunoprecipitation - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
  • Flow Cytometry - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

Key features and details

  • Mouse monoclonal [2C11] to SQSTM1 / p62 - BSA and Azide free
  • Suitable for: IHC-P, IP, WB, ICC/IF, Flow Cyt
  • Knockout validated
  • Reacts with: Human
  • Isotype: IgG2a

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Overview

  • Product name

    Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free
    See all SQSTM1 / p62 primary antibodies
  • Description

    Mouse monoclonal [2C11] to SQSTM1 / p62 - BSA and Azide free
  • Host species

    Mouse
  • Tested applications

    Suitable for: IHC-P, IP, WB, ICC/IF, Flow Cytmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant full length protein corresponding to Human SQSTM1/ p62 aa 1-440.
    Database link: Q13501

  • Positive control

    • WB: HeLa, Hap1, U-2 OS cell lysates. Flow Cyt: HeLa. IHC-P: Human lymph node. ICC/IF: HeLa and U-2 OS cells. IP: U-2 OS cell lysate
  • General notes

    This product was changed from ascites to tissue culture supernatant on 28th May 2019. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

    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. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.40
    Constituent: PBS
  • Carrier free

    Yes
  • Concentration information loading...
  • Purity

    Tissue culture supernatant
  • Clonality

    Monoclonal
  • Clone number

    2C11
  • Isotype

    IgG2a
  • Light chain type

    kappa
  • 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-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Isotype control

    • Mouse IgG2a, kappa monoclonal [MG2a-53] - Isotype control (ab18415)
  • KO cell lines

    • Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
  • KO cell lysates

    • Human SQSTM1 (p62) knockout HEK-293T cell lysate (ab263770)
  • KO cell pellets

    • Human SQSTM1 (p62) knockout HEK-293T cell pellet (ab278927)
  • 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 ab56416 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
IHC-P (3)
Use at an assay dependent concentration.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

IP (1)
Use at an assay dependent concentration.
WB (23)
Use at an assay dependent concentration.
ICC/IF (4)
Use at an assay dependent concentration.

Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS). Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

Flow Cyt
Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody

Notes
IHC-P
Use at an assay dependent concentration.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

IP
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.

Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS). Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

Flow Cyt
Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody

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
    • Omim: 601530 Human
    • SwissProt: Q13501 Human
    • Unigene: 709030 Human
    • 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 [2C11] - BSA and Azide free (ab56416)
      Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      All lanes : Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416) at 1/1000 dilution

      Lane 1 : Wild-type HEK293T cell lysate
      Lane 2 : SQSTM1 knockout HEK293T cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Observed band size: 62 kDa why is the actual band size different from the predicted?



      Lanes 1- 2: Merged signal (red and green). Green - ab56416 observed at 62 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

       ab56416 was shown to react with SQSTM1/p62 in wild-type HEK293T cells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate ab263770) was used. Wild-type HEK293T and SQSTM1 knockout HEK293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab56416 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      ab56416 was shown to react with SQSTM1 in wild-type U-2 OS cells in Immunocytochemistry with loss of signal observed in a SQSTM1 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1:1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1/10 000. The cells were then incubated with ab56416 at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-mouse secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.ing knock out cell lines.
    • Immunoprecipitation - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Immunoprecipitation - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Immunoprecipitation of SQSTM1 in U-2 OS cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab56416 pre-coupled to prot.G-Sepharose beads. Samples were washed and processed for western blot with ab207305 at 1/10,000. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
    • Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Western blot - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      All lanes : Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416) at 1/1000 dilution

      Lane 1 : Wild-type U-2 OS cell lysate
      Lane 2 : SQSTM1 knockout U-2 OS cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.


      ab56416 was shown to react with SQSTM1 in wild-type U-2 OS cells in Western blot with loss of signal observed in a SQSTM1 knockout cell line. Wild-type U-2 OS and SQSTM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab56416 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-mouse HRP secondary antibodies at 0.3ug/mL before imaging. This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knockout cell lines.

    • Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

      Monoclonal antibody to SQSTM1 (ab56416) on HeLa cell, antibody concentration 10 ug/ml.

      This image was generated using the ascites version of the product.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

      ab56416 (1µg/ml) staining SQSTM1 in human lymph node using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear and cytoplasmic staining.
      Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

      This image was generated using the ascites version of the product.

    • Flow Cytometry - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)
      Flow Cytometry - Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

      Overlay histogram showing HeLa cells stained with ab56416 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56416, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

      This image was generated using the ascites version of the product.

    Protocols

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

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet download

      Download

    References (731)

    Publishing research using ab56416? Please let us know so that we can cite the reference in this datasheet.

    ab56416 has been referenced in 731 publications.

    • Liu Y  et al. Paeonol inhibits apoptosis of vascular smooth muscle cells via up-regulation of autophagy by activating class III PI3K/Beclin-1 signaling pathway. Life Sci 264:118714 (2021). PubMed: 33157088
    • Chen Y  et al. m6A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells. Autophagy 17:457-475 (2021). PubMed: 31983283
    • Zimmermann C  et al. Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release. Autophagy 17:779-795 (2021). PubMed: 32079454
    • Liu M  et al. KIF5A-dependent axonal transport deficiency disrupts autophagic flux in trimethyltin chloride-induced neurotoxicity. Autophagy 17:903-924 (2021). PubMed: 32160081
    • Kim HK  et al. TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium. Autophagy 17:761-778 (2021). PubMed: 32167007
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-4 of 4 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-SQSTM1 / p62 antibody - Autophagosome Marker

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (CAFs)
    Permeabilization
    Yes - Tween-20 (1%)
    Specification
    CAFs
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    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 Jul 07 2020

    Immunocytochemistry/ Immunofluorescence abreview for Anti-SQSTM1 / p62 antibody - Autophagosome Marker

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (MRC5)
    Permeabilization
    Yes - 0.1% v/v Triton X-100 pH 7.4 for 5 min at RT
    Specification
    MRC5
    Blocking step
    BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    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.

    DR. Dimitra Kalamida

    Verified customer

    Submitted Nov 21 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-SQSTM1 / p62 antibody - Autophagosome Marker

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Skeletal Muscle)
    Permeabilization
    No
    Specification
    Skeletal Muscle
    Blocking step
    Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 22°C
    Fixative
    Methacarn
    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 Oct 01 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-SQSTM1 / p62 antibody - Autophagosome Marker

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (A431 epidermoid cancer cells)
    Permeabilization
    Yes - triton x-100
    Specification
    A431 epidermoid cancer cells
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
    Fixative
    Formaldehyde
    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.

    Dr. Alejandro Vazquez-Martin

    Verified customer

    Submitted Dec 02 2009

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