Flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized human peripheral blood mononuclear cells (PBMC) labeling CD3 with ab11089 at 1/400 dilution (red) compared with a Mouse IgG, monoclonal Isotype Control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Mouse IgG ab150157 (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-CD3 antibody [CD3-12] (ab11089) at 1 µg/ml

Lane 1 : THP-1 (Human monocytic leukemia cell line) whole cell lysate (negative control)
Lane 2 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate (negative control)
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) 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-Rat at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 23.1 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?

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 ab11089 overnight at 4°C. Antibody binding was detected using Goat anti-Rat secondary at a 1:10000 dilution for 1hr at room temperature and then imaged.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CB3 with ab11089 at 1/200 dilution, followed by ready to use Goat Anti-rat IgG H&L (HRP polymer) (ab214882). Positive staining on rat spleen tissue is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, followed by ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214882). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD3 with ab11089 at 1/200 dilution, followed by ready to use Goat Anti-Rat IgG H&L (HRP polymer) (ab214882). Positive staining on mouse spleen tissue is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, followed by ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214882). Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

 

IHC image of CD3 staining in a formalin fixed, paraffin embedded human tonsil tissue. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab11089 at 1/250 dilution for 15 minutes at room temperature. A goat anti-rat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was 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.

IHC image of CD3 staining in a formalin fixed, paraffin embedded normal mouse lymph node tissue.

The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab11089 at 1/250 dilution for 15 minutes at room temperature. A goat anti-rat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was 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.

ab11089 staining CD3 in mouse lymph node tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with formaldehyde and blocked with 15% serum for 60 minutes at 20°C. Antigen retrieval was by heat mediation in sodium citrate (pH 6). Samples were incubated with primary antibody (1/250 in TBS) for 18 hours at 20°C. An Alexa Fluor^®conjugated goat anti-rat IgG polyclonal (1/400) was used as the secondary antibody.

See Abreview

All lanes : Anti-CD3 antibody [CD3-12] (ab11089) at 1/1000 dilution

Lane 1 : Mouse thymus tissue lysate
Lane 2 : Rat thymus tissue lysate
Lane 3 : Human thymus tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution

Predicted band size: 23.1 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure: Lanes 1/3: 5 secs; Lanes 2: 3 secs.

All lanes : Anti-CD3 antibody [CD3-12] (ab11089) at 1/1000 dilution

Lane 1 : Jurkat (human T cell leukemia T lymphocyte)
Lane 2 : MOLT-4 (human lymphoblastic leukemia T lymphoblast)
Lane 3 : EL4 (mouse lymphoma T lymphocyte)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution

Predicted band size: 23.1 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 5 secs.

Anti-CD3 antibody [CD3-12] (ab11089) + Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Secondary
HRP-conjugated goat anti-rat IgG

Predicted band size: 23.1 kDa