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.

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 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.

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.

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

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

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.

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.