All lanes : Anti-CD3D antibody [EP4426] (ab109531) at 1/1000 dilution

Lane 1 : THP1 whole cell lysate (-ve control)
Lane 2 : Raji whole cell lysate (-ve control)
Lane 3 : Jurkat whole cell lysate
Lane 4 : Human Thymus tissue lysate
Lane 5 : Mouse Thymus tissue lysate
Lane 6 : Rat Thymus tissue lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab109531 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

This blot was produced using a 4-12% Bis-tris gel under the MES 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 Licor blocking buffer before being incubated with ab109531 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

IHC image of CD3D staining in a formalin fixed, paraffin embedded human B-Cell lymphoma tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab109531, 1/200 dilution, 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

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling CD3D with purified ab109531 at 1/500 dilution (4.5 µg/mL). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

Purified ab109531 at 1/50 dilution (2µg) immunoprecipitating CD3D in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab109531 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109531 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 23 kDa

IHC image of CD3D staining in a formalin fixed, paraffin embedded normal human tonsil tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab109531, 1/1000 dilution, 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

ab109531, at 1/100 dilution, staining paraffin embedded Human tonsil tissue by Immunohistochemistry.

Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

All lanes : Anti-CD3D antibody [EP4426] (ab109531) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HuT 78 cell lysate
Lane 3 : Human thymus tissue lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 19 kDa