Anti-TOMM20 antibody [4F3] - BSA and Azide free (ab56783)

Paraffin embedded human small intestine tissue stained for TOMM20 using ab56783 at 3 µg/ml in immunohistochemical analysis.

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Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling TOMM20 with ab56783 at 10 µg/ml. Cells are fixed with 4% PFA and permeabilized on ice in PBS 0.1% Triton. Samples were incubated with primary antibody at 4^oC overnight and fluorscein- conjugated secondary antibody at 4^oC  for 1 hour.  

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ab56783 staining TOMM20 in human keratinocytes by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with acetone and blocked with 1% BSA for 1 hour at 24°C. Samples were incubated with primary antibody (1/2000 in PBS + 1% BSA) for 24 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-mouse IgG polyclonal was used as the secondary antibody (1/5000).

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ab56783 staining TOMM20 in HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with PBS + 0.1% Triton X-100 and blocked with PBS+ 0.5% BSA + 0.2% fish skin gelatin for 1 hour at 25°C. Samples were incubated with primary antibody (1/2000 in PBS + 1% BSA) for 24 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-mouse IgG polyclonal was used as the secondary antibody (1/500).

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TOMM20 was immunoprecipitated using 0.5 mg HepG2 whole cell extract, 5 µg of Mouse monoclonal to TOMM20 (ab56783) and 50 µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10 min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 min under agitation.
Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 min at 70°C; 10 µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab56783.
Secondary: Protein G-HRP at 1/500 dilution.
Band: 14kDa: TOMM20. Non specific - 25kDa: We are unsure as to the identity of this extra band.

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Overlay histogram showing HeLa cells stained with ab56783 (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 (ab56783, 1 μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was a mix of mouse IgG1 [ICIGG1], (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4%PFA/permeabilized in 0.1% PBS-Tween used under the same conditions.

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