Anti-Dcp1a antibody (ab47811)

All lanes : Anti-Dcp1a antibody (ab47811) at 1 µg/ml

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : DCP1A knockout HEK-293 whole cell lysate
Lane 3 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 63 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab47811 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab47811 was shown to recognize DCP1A in wild-type HEK-293 cells as signal was lost at the expected MW in DCP1A  knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and DCP1A  knockout samples were subjected to SDS-PAGE. Ab47811 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/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.

IHC image of Dcp1a antibody staining in a section of formalin-fixed paraffin-embedded normal human placenta* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab47811, 5ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-Dcp1a antibody (ab47811) at 1 mg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) WCL at 10 µg
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) WCL
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) WCL

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 63 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 120 kDa (possible non-specific binding), 90 kDa (possible non-specific binding)


Exposure time: 12 minutes

This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab47811 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

Dcp1a was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to Dcp1a 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 ab47811.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 75kDa: Dcp1a.