Name: Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
SKU: ab109012
Rating: 5 (15 reviews)

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker is a rabbit recombinant monoclonal antibody that is used to detect SQSTM1 / p62 in Flow cytometry (Intra), ICC/IF, IP, Western blot. Suitable for Human, Mouse, Rat samples.

- Recombinant format for unrivaled batch-batch consistency
- Specificity confirmed with SQSTM1 knockout cell line validation
- Cited in over 400 publications

Related conjugates and formulations

ab203429
Conjugated
Alexa Fluor® 594 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab203430
Conjugated
Alexa Fluor® 555 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab225094
Conjugated
PE Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab225093
Conjugated
APC Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab185015
Conjugated
Alexa Fluor® 488 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab194720
Conjugated
HRP Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker
ab219581
Carrier free
Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free
ab194721
Conjugated
Alexa Fluor® 647 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker

Images

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (AB109012), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Abcam Recommends

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	WB	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested
Mouse	Expected	Tested	Expected	Expected
Rat	Expected	Tested	Expected	Expected

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/10000.00000 - 1/50000.00000	Notes -
Species Rat	Dilution info 1/10000.00000 - 1/50000.00000	Notes -
Species Human	Dilution info 1/10000.00000 - 1/50000.00000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1 µg/mL	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/10.00000 - 1/100.00000	Notes ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Associated Products

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Target data

See full target information SQSTM1

Function

The protein expressed by the SQSTM1 gene is an autophagy receptor essential for selective macroautophagy (aggrephagy). It acts as a bridge between polyubiquitinated cargo and autophagosomes, interacting with both the cargo and an autophagy modifier from the MAP1 LC3 family. Alongside WDFY3, it plays a role in forming and autophagically degrading cytoplasmic ubiquitin-containing inclusions and recruiting ubiquitinated proteins to nuclear PML bodies. SQSTM1 may regulate NFKB1 activation by TNF-alpha, nerve growth factor (NGF), and interleukin-1, and may influence titin/TTN signaling in muscle cells. It may also affect signaling cascades via ubiquitination and be involved in cell differentiation, apoptosis, immune response, and regulation of potassium channels. The protein is involved in endosome organization by retaining vesicles in the perinuclear cloud; ubiquitination by RNF26 allows it to attract vesicle-associated adapters, forming a molecular bridge to organize endosomal pathways. Additionally, it promotes the relocalization of Lys-63-linked ubiquitinated STING1 to autophagosomes and acts as an activator of the NFE2L2/NRF2 pathway by interacting with KEAP1, which inactivates the BCR(KEAP1) complex, resulting in nuclear accumulation of NFE2L2/NRF2 and expression of cytoprotective genes. This supplementary information is collated from multiple sources and compiled automatically.

Alternative names

ORCA, OSIL, SQSTM1, Sequestosome-1, EBI3-associated protein of 60 kDa, Phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa, Ubiquitin-binding protein p62, EBIAP, p60, p62

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR4844
Purification technique: Affinity purification Protein A
Specificity: This antibody got too weak band in rat tissues, you may need to optimize experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to get visible band. However, it performs very well in rat cell lines.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Stable for 12 months at -20°C

Notes

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), ICC/IF, IP and WB.

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) was first used in a scientific publication in 2014 and has been cited over 445 times in peer reviewed journals. It's performance in Western Blot in human, mouse and rat samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) has been confirmed by Western Blot testing in SQSTM1 / p62 knockout HEK-293T cells (Human SQSTM1 (p62) knockout HEK-293T cell lysate ab263770).

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) has 15 independent reviews from customers.

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) specifically detects SQSTM1 / p62 (UniProt ID: Q13501; Molecular weight: 48kDa) and is sold in 100 µL and 1 mL selling sizes.

Conjugation-ready, carrier free format available for antibody clone EPR4844 - Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free ab219581.

Antibody clone EPR4844 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 488, HRP, Alexa Fluor® 647, Alexa Fluor® 594, Alexa Fluor® 555, APC, PE (Alexa Fluor® 488 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab185015, HRP Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab194720, Alexa Fluor® 647 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab194721, Alexa Fluor® 594 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab203429, Alexa Fluor® 555 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab203430, APC Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab225093, PE Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab225094).

NEW: Explore our alternative host species for clone [EPR4844] : goat. SQSTM1, also known as p62, is a multifunctional scaffolding protein widely used in neuro research for its role in autophagy and protein degradation. It is upregulated in several neurodegenerative disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Researchers use SQSTM1/p62 to study the mechanisms of protein aggregation and neuroinflammation, making it essential for understanding the progression of neurodegenerative diseases and identifying potential therapeutic targets.

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
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15 product images

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1 - 4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
ab109012 was shown to specifically react with SQSTM1 in wild-type HAP1 cells. No band was observed when SQSTM1 knockout samples were used. Wild-type and SQSTM1 knockout samples were subjected to SDS-PAGE, ab109012 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/10000 dilution
Lane 1: Wild-type HAP1 whole cell lysate at 20 µg
Lane 2: SQSTM1 knockout HAP1 whole cell lysate at 20 µg
Lane 3: HeLa whole cell lysate at 20 µg
Lane 4: HepG2 whole cell lysate at 20 µg
Predicted band size: 47 kDa
Immunoprecipitation - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Immunoprecipitation of SQSTM1 in U-2 OS cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab109012 pre-coupled to prot.A-Sepharose beads. Samples were washed and processed for western blot with SQSTM1 / P62 antibody at 1/5000. 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.
All lanes: Immunoprecipitation - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Predicted band size: 47 kDa
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1-4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
ab109012 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in knockout cell line Human SQSTM1 (p62) knockout HCT116 cell line ab266871 (knockout cell lysate Human SQSTM1 (p62) knockout HCT116 cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lane 1: Wild-type HCT116 cell lysate at 20 µg
Lane 2: SQSTM1 knockout HCT116 cell lysate at 20 µg
Lane 3: HepG2 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
ab109012 staining SQSTM1 in wild-type Hap1 cells and SQSTM1 knockout Hap1 cells treated with chloroquine (Chloroquine diphosphate, apoptosis and autophagy inhibitor ab142116, 50μM for 24 hrs). The cells were fixed with 100% methanol (5 min) or with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109012 at 0.1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Residual signal is observed in KO cells when paraformaldehyde is used for fixation, therefore we recommend using methanol fixation with this antibody. Alternatively, please use Anti-SQSTM1 / p62 antibody [EPR23101-103] - Autophagosome Marker ab240635 or Anti-SQSTM1 / p62 antibody [EPR18351] - Autophagosome Marker ab207305 which have been KO validated in both paraformaldehyde and methanol fixed cells.
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
ab109012 staining SQSTM1/p62 (autophagosome) in control HeLa cells (left panel) and SQSTM1/p62 in HeLa cells treated with 1uM bafilomycin A1 (Bafilomycin A1, (V)-ATPase inhibitor ab120497) for 18hrs (right panel). The cells were fixed with methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109012 at 5ug/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1- 2: Merged signal (red and green). Green - ab109012 observed at 64 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
ab109012 was shown to react with SQSTM1/p62 in wild-type HEK293T cells in western blot. Loss of signal was observed when knockout cell line Human SQSTM1 (p62) knockout HEK-293T cell line ab255343 (knockout cell lysate Human SQSTM1 (p62) knockout HEK-293T 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. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/10000 dilution
Lane 1: Wild-type HEK293T cell lysate at 20 µg
Lane 2: SQSTM1 knockout HEK293T cell lysate at 20 µg
Lane 2: Western blot - Human SQSTM1 (p62) knockout HEK-293T cell line (Human SQSTM1 (p62) knockout HEK-293T cell line ab255343)
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 64 kDa
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Different batches of ab109012 were tested on MCF7 (Human breast adenocarcinoma epithelial cell) lysate at 0.4 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 62 kDa.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Predicted band size: 47 kDa
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lanes 1-4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
ab109012 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in CRISPR/Cas9 edited cell line Human SQSTM1 (p62) knockout HCT116 cell line ab266871 (CRISPR/Cas9 edited cell lysate Human SQSTM1 (p62) knockout HCT116 cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 CRISPR/Cas9 edited samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lane 1: Wild-type HCT116 cell lysate at 20 µg
Lane 2: SQSTM1 CRISPR/Cas9 edited HCT116 cell lysate at 20 µg
Lane 2: Western blot - Human SQSTM1 (p62) knockout HCT116 cell line (Human SQSTM1 (p62) knockout HCT116 cell line ab266871)
Lane 3: HepG2 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
ab109012 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 ab109012 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2ug/mL before imaging. 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.
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/10000 dilution
Lane 1: Wild-type U-2 OS cell lysate at 20 µg
Lane 2: SQSTM1 knockout U-2 OS cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 47 kDa
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Purified ab109012 staining SQSTM1 in wild-type HAP1 cells (top panel) and SQSTM1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109012 at 1μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SQSTM1 / p62 with purified ab109012 at 1/50 dilution (10 μg/ml) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/1000 dilution
Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Lane 2: Mouse brain lysates at 20 µg
Lane 3: rat brain lysates at 20 µg
Lane 4: Mouse lung lysates at 20 µg
Lane 5: Rat lung lysates at 20 µg
Lane 6: Mouse heart lysates at 20 µg
Lane 7: Rat heart lysates at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 47 kDa
Observed band size: 62 kDa
Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
All lanes: Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lane 1: MCF-7 at 10 µg
Lane 2: HeLa at 10 µg
Lane 3: SKBR-3 at 10 µg
Lane 4: 293T at 10 µg
Predicted band size: 47 kDa
Observed band size: 62 kDa
Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Overlay histogram showing HeLa cells stained with unpurifiedab109012 (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 (ab109012, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Immunocytochemistry/ Immunofluorescence - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Unpurified ab109012 staining SQSTM1/p62 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab109012 at 1μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in pseudocolor red) followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).