Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells fixed in 4% PFA and stained with purified ab40763 at a dilution of 1 in 20 (red line).

The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Immunohistochemical staining of paraffin embedded human tonsil with purified ab40763 at a working dilution of 1/250.

The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (shown in the inset).

Immunofluorescence staining of Jurkat cells (Human T cell leukemia cell line from peripheral blood) with purified ab40763 at a working dilution of 1/100, counterstained with DAPI.

The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel.

The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab40763 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (purified) + HuT-78 cell lysate at 20 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 147 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

Unpurified ab40763 showing positive staining in normal colon lymphoid cells tissue.

Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (purified) + Raji (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 147 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

Unpurified ab40763 showing positive staining in normal spleen tissue.

Unpurified ab40763 showing negative staining in skeletal muscle tissue.

Unpurified ab40763 showing negative staining in normal kidney tissue.

Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution (unpurified, blocked with 3% milk) + Jurkat (Human T cell lymphoblast-like cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 147 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 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 ab40763 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Equilibrium disassociation constant (KD)
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