Anti-RAB27A antibody (ab55667)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAB27A antibody (ab55667)

RAB27A antibody (ab55667) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human lymphoma. Heat induced antigen retrieval with Citrate buffer pH 6 has been performed prior to immunostaining.

Immunocytochemistry/ Immunofluorescence - RAB27A antibody (ab55667)

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).

This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic

Western blot - RAB27A antibody (ab55667)

All lanes : Anti-RAB27A antibody (ab55667) at 1/200 dilution

Lane 1 : whole cell lysate of HEK 293 transfected with empty pCDNA3 vector
Lane 2 : whole cell lysate of HEK 293 cells expressing Rab27a-GFP

Lysates/proteins at 20 µg per lane.

Secondary
HRP conjugated goat anti-mouse
developed using the ECL technique

Predicted band size : 25 kDa
Observed band size : 52 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 minutes

Rab27a (25kDa)+GFP(27kDa)=52kDa

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.

This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic

Immunoprecipitation - RAB27A antibody (ab55667)

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

This image is courtesy of an Abreview submitted by Dr Vladimir Milenkovic