Anti-SQSTM1 / p62 antibody [EPR18351] - BSA and Azide free (ab227992)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18351] to SQSTM1 / p62 - BSA and Azide free
- Suitable for: ICC, IHC-P, WB, IP, Flow Cyt
- Knockout validated
- Reacts with: Human
Overview
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Product name
Anti-SQSTM1 / p62 antibody [EPR18351] - BSA and Azide free
See all SQSTM1 / p62 primary antibodies -
Description
Rabbit monoclonal [EPR18351] to SQSTM1 / p62 - BSA and Azide free -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species Flow Cyt HumanIHC-P HumanIP HumanWB Human -
Immunogen
Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Human liver and kidney lysates; HCT116, HepG2 and HeLa whole cell lysates. IHC-P: Human hepatocellular carcinoma and lung cancer tissues. ICC/IF: HeLa cells (untreated and treated with chloroquine), HAP1 cells (untreated and treated with chloroquine) Flow Cyt: PC-3 cells. IP: HeLa and HepG2 whole cell lysates.
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General notes
Ab227992 is the carrier-free version of ab207305. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.
Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with
ab227992 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.
Maxpar® is a trademark of Fluidigm Canada Inc.
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 the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.2
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18351 -
Isotype
IgG -
Research areas
Images
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All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?This data was developed using ab207305, the same antibody clone in a different buffer formulation.
Lanes 1-4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.
ab207305 Anti-SQSTM1 / p62 antibody [EPR18351] was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in knockout cell line ab266871 (knockout 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. ab207305 and 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 (ab216773) and 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.
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ab207305 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 ab207305 at 1μg/ml and 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) (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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
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SQSTM1 / p62 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab207305 at 1/40 dilution.
Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10ug (Input).
Lane 2: ab207305 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
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Clone EPR18351 (ab227992) has been successfully conjugated by Abcam. This image was generated using Anti-SQSTM1 / p62 antibody [EPR18351] (PE). Please refer to ab225454 for protocol details.
Overlay histogram showing HeLa cells untreated (red line) and HeLa cells treated with Chloroquine, 50µM, 24 hours, (green line) stained with ab225454. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225454, 1/5000 dilution) for 30 min at 22°C.
Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.
This antibody gave a positive signal in HeLa +/- Chloroquine cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.
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Clone EPR18351 (ab227992) has been successfully conjugated by Abcam. This image was generated using Anti-SQSTM1 / p62 antibody [EPR18351] (Alexa Fluor® 647). Please refer to ab225453 for protocol details.
ab225453 staining SQSTM1 / p62 in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (50μM, 24 hours). The cells were fixed with 4% formaldehyde (10min), 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 ab225453 at 5μg/ml (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 488), at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm and weakly nucleus staining on human lung cancer is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin-embedded human liver tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on Hhman normal liver tissue. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Flow cytometric analysis of 4% paraformaldehyde-fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling SQSTM1 / p62 with ab207305 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
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SQSTM1 / p62 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab207305 at 1/40 dilution.
Western blot was performed from the immunoprecipitate using ab207305 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HepG2 whole cell lysate 10ug (Input).
Lane 2: ab207305 IP in HepG2 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab207305 in HepG2 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab207305).
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This ICC data was generated using the same anti-SQSTM1/p62 antibody clone, EPR18351, in a different buffer formulation (cat# ab207305).
ab207305 staining SQSTM1 in wild-type HAP1 cells and knockout cells, untreated and chloroquine-treated (50μM, 24 hours). The cells were fixed with 4% formaldehyde (10min), 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 ab207305 at 1μg/ml and 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) (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).
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This IHC data was generated using the same anti-SQSTM1/p62 antibody clone, EPR18351, in a different buffer formulation (cat# ab207305).
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling SQSTM1 / p62 with ab207305 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on human hepatocellular carcinoma is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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