This antibody gave a positive signal in HeLa, SHSY-5Y and MALME-3M whole cell lysates, and in the following tissue lysates: Human Cortex, Rat Cortex, Rat Hippocampus, Human Melanoma.
Flow Cytometry: SHSY-5Y cells
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Western blot - Anti-Drebrin antibody [mAbcam60932] (ab60932)
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: Drebrin knockout HAP1 whole cell lysate (20 µg) Lane 3: SH-SY5Y whole cell lysate (20 µg)
Lanes 1 - 3: Merged signal (red and green). Green - ab60932 observed at 120 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab60932 was shown to specifically react with Drebrin in wild-type cells as signal was lost in Drebrin knockout cells. Wild-type and Drebrin knockout samples were subjected to SDS-PAGE. Ab60932 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 µg/ml and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Drebrin antibody [mAbcam60932] (ab60932)This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom
IHC-P image of mouse SVZ sections showing Drebrin staining using ab60932 (1/2000). The sections were fixed using Formaldehyde. Heat mediated antigen retrieval was performed using citric acid at pH6. The sections were blocked using 1% BSA for 10 mins at 21°C.
Ab60932 detects a band around 110-kDa. While this differs to its predicted molecular weight of 71-kDa, it is consistent with what has been described in the literature(PMID:10604653).
Western blot - Drebrin antibody [mAbcam60932] (ab60932)
All lanes : Anti-Drebrin antibody [mAbcam60932] (ab60932) at 10 µg/ml
Lane 1 : Human Cortex Neuronal cell lysate
Lane 2 : Rat Cortex Neuronal cell lysate Lane 3 : Rat Hippocampus Tissue Lysate Lane 4 : Human skin tumor tissue lysate (melanoma) - total protein (ab29339) Lane 5 : Malme 3M (Human melanoma cells) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution
Overlay histogram showing SHSY-5Y cells stained with ab60932 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab60932, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SHSY-5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Kaczmarski W et al. Intracellular distribution of differentially phosphorylated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). J Neurosci Res92:162-73 (2014).
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