Immunocytochemistry/ Immunofluorescence - Dcp2 antibody (ab28658)

ICC/IF image of ab28658 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab28658, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Dcp2 antibody (ab28658)

Image courtesy of Human Protein Atlas

ab28658 stainning Dcp2 in paraffin embedded human kidney. Kidney tissue sections (4 µm) were incubated with ab28658 (1/35 dilution) for 30 minutes at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab28658 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

Western blot - Dcp2 antibody (ab28658)

All lanes : Anti-Dcp2 antibody (ab28658) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Dcp2 peptide (ab30458) at 1 µg/ml
Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Dcp2 peptide (ab30458) at 1 µg/ml
Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Dcp2 peptide (ab30458) at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 48.4 kDa
Observed band size : 52,58 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 : 10 minutes

Dcp2 contains a number of potential phosphorylation sites (Swiss Prot data) which may explain its migration at a higher molecular weight than predicted.

Immunoprecipitation - Anti-Dcp2 antibody (ab28658)

Dcp2 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to Dcp2 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, Jurkat 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 ab28658.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 52,58kDa: Dcp2.