Anti-CRBN antibody [4D6]

• WB

Lab

Western blot - Anti-CRBN antibody [4D6] (AB244223)

Lanes 1 - 4 : Merged signal (red and green). Green - ab244223 observed at 55 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab244223 was shown to react with CRBN in MOLT4 (Licensor sample) wild-type cells in Western blot. Loss of signal was observed when CRBN knockout sample was used. MOLT4 (Licensor sample) wild-type and CRBN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab244223 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 2 μg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CRBN antibody [4D6] (ab244223) at 2 µg/mL

Lane 1:

wild-type MOLT4 (Licensor sample) cell lysate at 20 µg

Lane 2:

CRBN knockout MOLT4 cell lysate at 20 µg

Lane 3:

NIH/3T3 cell lysate at 20 µg

Lane 4:

MEF-1 cell lysate at 20 µg

Predicted band size: 51 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-CRBN antibody [4D6] (AB244223)

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 5% milk before ab244223 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at a 2ug/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CRBN antibody [4D6] (ab244223) at 2 µg/mL

Lane 1:

MOLT-4 whole cell lysate at 20 µg

Lane 2:

HEK293 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

SH-SY5Y whole cell lysate at 20 µg

Predicted band size: 51 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-CRBN antibody [4D6] (AB244223)

Anti-CRBN antibody [4D6] (ab244223) staining at 2 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab244223 was shown to bind specifically to CRBN. A band was observed at 51 kDa in wild-type cell lysates with no signal observed at this size in CRBN knockout cell lines. To generate this image, wild-type and CRBN knockout cell lysates were analysed : CRBN knockout A549 cell line (ab288959), CRBN knockout HCT 116 cell line (ab288959) and CRBN knockout MOLT-4. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CRBN antibody [4D6] (ab244223) at 2 µg/mL

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CRBN knockout A549 cell line (ab288959)

Lane 2:

CRBN knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HCT 116 cell lysate at 20 µg

Lane 4:

Western blot - Human CRBN knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-crbn-knockout-hct116-cell-line-ab289186'>ab289186</a>)

Lane 4:

CRBN knockout HCT 116 cell lysate at 20 µg

Lane 5:

Wild-type MOLT-4 cell lysate at 20 µg

Lane 6:

CRBN knockout MOLT-4 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Predicted band size: 51 kDa

Observed band size: 51 kDa

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• WB

Lab

Western blot - Anti-CRBN antibody [4D6] (AB244223)

Western blot : Anti-CRBN antibody [4D6] (ab244223) staining at 0.2 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab244223 was shown to bind specifically to CRBN. A band was observed at 51 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CRBN knockout cell line. To generate this image, wild-type and CRBN knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CRBN antibody [4D6] (ab244223) at 0.2 µg/mL

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

CRBN knockout MCF7 cell lysate at 20 µg

Lane 3:

Wild-type A549 ab288558 cell lysate at 20 µg

Lane 4:

CRBN knockout A549 ab288959 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 51 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CRBN antibody [4D6] (AB244223)

IHC image of CRBN staining in a section of formalin-fixed paraffin-embedded normal human testis* performed on a Leica BOND™ system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab244223, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.