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Our Abpromise guarantee covers the use of ab55667 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 25 kDa.|
|IHC-P||Use a concentration of 3 µg/ml.|
|IP||Use at an assay dependent concentration. See Abreview.|
|IHC-FoFr||Use at an assay dependent concentration. PubMed: 21832089|
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: RAB27A knockout HAP1 whole cell lysate (20 µg)
Lane 3: Jurkat whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab55667 observed at 27 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab55667 was shown to specifically react with RAB27A when RAB27A knockout samples were used. Wild-type and RAB27A knockout samples were subjected to SDS-PAGE. Ab55667 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 2.5 ug/ml and 1/10000 dilution respectively. Blots were developed with IRDye® 800CW Goat anti-Mouse IgG (H + L) and IRDye® 680 Goat anti-Rabbit IgG (H + L) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Immunocytochemistry/ Immunofluorescence analysis of SK-MEL-28 cells labeling RAB27A with ab55667 at 1/200 dilution. The cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton-X in PBS. A polyclonal goat anti-mouse IgG Alexa Fluor® 488 secondary antibody was used at 1/1000 dilution.
GAPDH (36.1 kDa) used as specificity and loading control.
ab55667 staining cow Retinal Pigment Epithelium (RPE) cells by ICC/IF. Cells were PFA fixed and permeabilized in 0.5% Triton X-100 prior to blocking in 10% serum for 20 minutes at 25°C. The primary antibody was diluted 1/200 and incubated with the sample for 12 hours at 4°C. An Alexa Fluor® 546 conjugated goat anti-mouse antibody was used as the secondary. Rab27a, which localizes to the apical site of the RPE cells, is labeled with red fluorescence. Nuclei were counterstained with DAPI (blue).
The blot was blocked with 5% milk for 30 minutes at 25°C and incubated with the primary antibody for 12 hours at 4°C.
ab55667 (undiluted) was incubated with whole cell lysate of HEK 293 cells transfected with Rab27a-GFP and a Protein G matrix for 12 hours at 4°C to achieve immunoprecipitation. 20µg of lysate were present in the input.
ab55667 (diluted 1/200) was also used in the western blotting step.
Lane 1. lysate of HEK 293 cells expressing Rab27a-GFP
Lane 2. IP with ab55667
Lane 3. Not bound fraction
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