Recombinant Anti-CD45 antibody [EP322Y] (ab40763)

False colour image of Western blot: Anti-CD45 antibody [EP322Y] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40763 was shown to bind specifically to CD45. A band was observed at 160-220 kDa in wild-type Jurkat cell lysates with no signal observed at this size in PTPRC CRISPR-Cas9 edited cell line ab274932 (CRISPR-Cas9 edited cell lysate ab274990). The band observed in the CRISPR-Cas9 edited lysate lane below 160-220 kDa is likely to represent a truncated form of CD45. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and PTPRC CRISPR-Cas9 edited Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1% Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 2ug/ml dilution.

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.

Blocking/Dilution buffer: 5% NFDM/TBST

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

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.

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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