Product datasheet

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome
Marker ab109012
リコンビナント

15 Abreviews 445 References 画像数 16
製品の概要
製品名

Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker

製品の詳細

Rabbit monoclonal [EPR4844] to SQSTM1 / p62 - Autophagosome Marker

由来種Rabbit

特異性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.

アプリケーション

適用あり : WB, Flow Cyt (Intra), IP, ICC/IF

種交差性

交差種 : Mouse, Rat, Human

免疫原

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

ポジティブ･コントロール

WB: U-2 OS, MCF-7, HAP1, HCT116, HeLa, HepG2, SKBR-3, MCF-7 and HEK-293T celllysates; Mouse and rat brain, heart and lung tissue lysates. ICC/IF: HeLa cells (untreated andtreated with chloroquine or bafilomycin) and HAP1 cells. Flow Cyt (intra): HeLa cells. IP: U-2 OScell lysate.

特記事項

We would like to recommend the following products as good alternatives to ab109012 that
perform well in Immunocytochemistry/ Immunofluorescence:ab240635 and ab207305
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 production
For more information see here.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit
monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

製品の特性
製品の状態

Liquid1保存方法Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C.
Stable for 12 months at -20°C.

バッファー

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA

精製度

Protein A purified

ポリ /モノモノクローナル

クローン名

EPR4844

アイソタイプ

IgG

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab109012の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション

WB

特記事項

Abreviews(9)Flow Cyt (Intra)IP

1/10000 - 1/50000. Detects a band of approximately 62 kDa.
1/10 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as anisotype control with this antibody.

(2)

ICC/IF

Use at an assay dependent concentration.
Use a concentration of 1 µg/ml.

ターゲット情報機能

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 inmuscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates theinteraction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation,apoptosis, immune response and regulation of K(+) channels.

組織特異性

Ubiquitously expressed.

関連疾患

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

配列類似性

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

ドメイン

The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinatedsubstrates. Mediates the interaction with TRIM55.
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The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5
and NBR1.
The ZZ-type zinc finger mediates the interaction with RIPK1.
翻訳後修飾

Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro byTTN.

細胞内局在

Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes tothe sarcomeric band (By similarity). Localizes to late endosomes. May also localize to thenucleus. 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 hepatocellularcarcinoma.

画像ab109012 staining SQSTM1 in wild-type Hap1 cells and SQSTM1
knockout Hap1 cells treated with chloroquine (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 X100 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 thenincubated with ab109012 at 0.1μg/ml concentration and ab7291(Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at
4°C followed by a further incubation at room temperature for 1h witha goat secondary antibody to rabbit IgG (Alexa Fluor® 488)
(ab150081) at 2 μg/ml (shown in green) and a goat secondary
Immunocytochemistry/ Immunofluorescence - Anti-

antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml

SQSTM1 / p62 antibody [EPR4844] -(shown in red). Nuclear DNA was labelled in blue with DAPI.

Autophagosome Marker (ab109012)

Image was taken with a confocal microscope (Leica-Microsystems
TCS SP8).
Residual signal is observed in KO cells when paraformaldehyde isused for fixation, therefore we recommend using methanol fixation
with this antibody. Alternatively, please use ab240635 or ab207305
which have been KO validated in both paraformaldehyde andmethanol fixed cells.

3All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012) at 1/10000 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: 64 kDa
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 (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 whenknockout cell line ab255343 (knockout cell lysate ab263770) wasused. Wild-type HEK293T and SQSTM1 knockout HEK293T celllysates 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(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 (ab216773) and Goat antiMouse IgG H&L (IRDye®680RD) preadsorbed (ab216776)
secondary antibodies at 1 in 20000 dilution for 1 hour at room
temperature before imaging.

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ab109012 staining SQSTM1/p62 (autophagosome) in control HeLacells (left panel) and SQSTM1/p62 in HeLa cells treated with 1uM
bafilomycin A1 (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 thenincubated with ab109012 at 5ug/ml and 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) (ab150081) at 2 μg/ml (shown in green) and agoat secondary antibody to mouse IgG (Alexa Fluor® 594)
Immunocytochemistry/ Immunofluorescence - Anti-(ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA

SQSTM1 / p62 antibody [EPR4844] -was labelled in blue with DAPI.

Autophagosome Marker (ab109012)

Image was taken with a confocal microscope (Leica-Microsystems,TCS SP8).

Immunoprecipitation of SQSTM1 in U2OSn cells. Lysates wereprepared and immunoprecipitation was performed using 1.0 μg ofab109012 pre-coupled to prot.A-Sepharose beads. Samples were
washed and processed for western blot with GTX629890 at 1/5000.
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 withYCharOS to support their mission of antibody characterisation
using knock out cell lines.

Immunoprecipitation - Anti-SQSTM1 / p62 antibody[EPR4844] - Autophagosome Marker (ab109012)

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Immunoprecipitation of SQSTM1 in U-2 OS cells. Lysates wereprepared 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% unboundfraction; IP=immunoprecipitate. These data were provided byYCharOS Inc., an open science company with the mission ofcharacterizing 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.
Immunoprecipitation - Anti-SQSTM1 / p62 antibody[EPR4844] - Autophagosome Marker (ab109012)

All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : SQSTM1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HepG2 whole cell lysate
Lysates/proteins at 20 µg per lane.

Western blot - Anti-SQSTM1 / p62 antibodyLanes 1 - 4: Merged signal (red and green). Green - ab109012[EPR4844] - Autophagosome Marker (ab109012)

observed at 55 kDa. Red - loading control, ab8245, observed at 37kDa.
ab109012 was shown to specifically react with SQSTM1 in wildtype 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 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®

6developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW)
preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye®
680RD) preabsorbed ab216776 secondary antibodies at 1/20,000
dilution for 1 hour at room temperature before imaging.

All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012)

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 CRISPR/Cas9 edited HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.

SecondaryWestern blot - Anti-SQSTM1 / p62 antibody

All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW)[EPR4844] - Autophagosome Marker (ab109012)

preadsorbed (ab216773) at 1/10000 dilution

Observed band size: 55 kDa

Lanes 1-4: Merged signal (red and green). Green - ab109012
observed at 55 kDa. Red - loading control ab8245 observed at 36kDa.
ab109012 Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker was shown to specifically react with
SQSTM1 / p62 in wild-type HCT116 cells. The band observed inCRISPR/Cas9 edited cell line ab266871 (CRISPR/Cas9 edited celllysate ab257052) lane below 55 kDa may represent truncatedforms and cleaved fragments. This has not been investigatedfurther. Wild-type and SQSTM1 / p62 CRISPR/Cas9
edited samples were subjected to SDS-PAGE. ab109012 andAnti-GAPDH antibody [6C5] - Loading Control (ab8245) wereincubated 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 (ab216773) and Goat antiMouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
secondary antibodies at 1 in 20000 dilution for 1 hour at room
temperature before imaging.

7All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012) at 1/10000 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.

Western blot - Anti-SQSTM1 / p62 antibody[EPR4844] - Autophagosome Marker (ab109012)

ab109012 was shown to react with SQSTM1 in wild-type U-2 OScells in Western blot with loss of signal observed in a SQSTM1knockout cell line. Wild-type U-2 OS and SQSTM1 knockout celllysates were subjected to SDS-PAGE. Membranes were blocked in
5% milk in TBST for 1 hr before incubation with ab109012 overnightat 4 °C at a 1/10000 dilution. Blots were incubated with goat antirabbit 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 byenabling the life science community to better evaluate commerciallyavailable antibodies.

Purified ab109012 staining SQSTM1 in wild-type HAP1 cells (toppanel) 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 at1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolourred) 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) (ab150081) at 2 μg/ml (shown in green).
Nuclear DNA was labelled in blue with DAPI.
Immunocytochemistry/ Immunofluorescence - Anti-Image was taken with a confocal microscope (Leica-Microsystems,SQSTM1 / p62 antibody [EPR4844] -TCS SP8).

Autophagosome Marker (ab109012)

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Intracellular Flow Cytometry analysis of HeLa (Human cervix
adenocarcinoma epithelial cell) cells labeling SQSTM1 / p62 withpurified 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, 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).

Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62
antibody [EPR4844] - Autophagosome Marker(ab109012)

All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012) at 1/1000 dilution (Purified)

Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell)whole cell lysates

Lane 2 : Mouse brain lysates
Lane 3 : rat brain lysates
Lane 4 : Mouse lung lysates
Lane 5 : Rat lung lysates
Lane 6 : Mouse heart lysates
Western blot - Anti-SQSTM1 / p62 antibody

Lane 7 : Rat heart lysates[EPR4844] - Autophagosome Marker (ab109012)

Lysates/proteins at 20 µg per lane.

SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000
dilution

Observed band size: 62 kDa

9Different batches of ab109012 were tested on MCF7 (Humanbreast adenocarcinoma epithelial cell) lysate at 0.4 µg/ml. 15 µg oflysate was loaded in each lane. Bands observed at 62 kDa.

Western blot - Anti-SQSTM1 / p62 antibody[EPR4844] - Autophagosome Marker (ab109012)

All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012) (Unpurified)

Lane 1 : MCF-7
Lane 2 : HeLa
Lane 3 : SKBR-3
Lane 4 : 293T
Lysates/proteins at 10 µg per lane.

Observed band size: 62 kDa
Western blot - Anti-SQSTM1 / p62 antibody[EPR4844] - Autophagosome Marker (ab109012)

10

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) (ab150077) at 1/2000 dilution for 30min at 22°C. Isotype control antibody (black line) was rabbit IgG
Flow Cytometry (Intracellular) - Anti-SQSTM1 / p62(monoclonal) (1µg/1x106 cells) used under the same conditions.

antibody [EPR4844] - Autophagosome Marker

Unlabelled sample (blue line) was also used as a control.

(ab109012)

Acquisition of >5,000 events were collected using a 20mW Argon
ion laser (488nm) and 525/30 bandpass filter.

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 5minutes and then blocked with 1% BSA/10% normal goat
serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were thenincubated overnight at +4°C with ab109012 at1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin (AlexaFluor® 594), at 1/250 dilution (shown in pseudocolor red) followed
by a further incubation at room temperature for 1h with a goatsecondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081)
at 2 μg/ml (shown in green). Nuclear DNA was labelled with DAPI
Immunocytochemistry/ Immunofluorescence - Anti-(shown in blue).

SQSTM1 / p62 antibody [EPR4844] -Image was taken with a confocal microscope (Leica-Microsystems,Autophagosome Marker (ab109012)

TCS SP8).

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Anti-SQSTM1 / p62 antibody [EPR4844] Autophagosome Marker (ab109012)

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