All lanes : Anti-CRBN antibody [4D6] (ab244223) at 2 µg/ml

Lane 1 : MOLT4 wild-type (Licensor sample) cell lysate
Lane 2 : CRBN knockout MOLT4 cell lysate
Lane 3 : NIH/3T3 cell lysate
Lane 4 : MEF-1 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 51 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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 : Anti-CRBN antibody [4D6] (ab244223) at 2 µg/ml

Lane 1 : MOLT-4 whole cell lysate
Lane 2 : HEK293 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : SH-SY5Y whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 51 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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.

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.