Anti-Drebrin antibody (ab60933)

ab60933 staining Drebrin in mouse brain tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using Citrate buffer pH6. Samples were then blocked with 1% BSA for 10 minutes at 21°C followed by incubation with the primary antibody at a 1/500 dilution, for 2 hours at 21°C. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as secondary antibody at a 1/200 dilution.

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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)
Lane 4: Hu cerebellum whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab60933 observed at 100120 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab60933 was shown to specifically react with Drebrin in wild-type HAP1 cells as signal was lost in Drebrin knockout cells. Wild-type and Drebrin knockout samples were subjected to SDS-PAGE. ab60933 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Drebrin antibody (ab60933) at 1 µg/ml

Lane 1 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
Lane 2 : SK N SH (Human neuroblastoma) Whole Cell Lysate
Lane 3 : SK N BE (Human neuroblastoma) Whole Cell Lysate
Lane 4 : Human Cortex Neuronal cell lysate
Lane 5 : Rat Cortex Neuronal cell lysate
Lane 6 : Mouse Cortex Neuronal cell lysate
Lane 7 : Rat Hippocampus Tissue Lysate
Lane 8 : Mouse Hippocampus Tissue Lysate
Lane 9 : Malme 3M (Human melanoma cells) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 100,120 kDa why is the actual band size different from the predicted?
Additional bands at: 35 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes


The banding pattern observed is consistent with what has been described in the literature (PMID:10234022, 8929425). Drebrin has two isoforms produced by alternative splicing.

Drebrin was immunoprecipitated using 0.5mg SHSY5Y whole cell extract, 5µg of Rabbit polyclonal to Drebrin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, SHSY5Y whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab60933.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 100kDa: Drebrin.

ICC/IF image of ab60933 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab60933, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) PC12 cells at 5µg/ml.