Recombinant Anti-CD45 antibody [EP322Y] - BSA and Azide free (ab214437)

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.

This data was developed using ab40763, the same antibody clone in a different buffer formulation.

This data was developed using ab40763, the same antibody clone in a different buffer formulation.

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 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). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763). 

Immunofluorescence staining of Jurkat cells with purified ab40763 at a working dilution of 1/100, counter-stained 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

Unpurified ab40763 showing negative staining in Normal kidney tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

Clone EP322Y (ab214437) has been successfully conjugated by Abcam. This image was generated using Anti-CD45 antibody [EP322Y] (Alexa Fluor^® 647). Please refer to ab200317 for protocol details. 

Overlay histogram showing Jurkat (human T cell leukemia cell line from peripheral blood) cells stained with ab200317 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab200317, 1/50000 dilution) for 30 min at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter. 

This antibody gave a positive signal in Jurkat cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions. 

Clone EP322Y (ab214437) has been successfully conjugated by Abcam. This image was generated using Anti-CD45 antibody [EP322Y] (PE). Please refer to ab214501 for protocol details. 

Overlay histogram showing Jurkat (human T cell leukemia cell line from peripheral blood) cells stained with ab214501 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab214501, 1/500 dilution) for 30 min at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter. 

This antibody gave a positive signal in Jurkat cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions. 

Clone EP322Y (ab214437) has been successfully conjugated by Abcam. This image was generated using Anti-CD45 antibody [EP322Y] (Alexa Fluor® 488). Please refer to ab200315 for protocol details.

ab200315 staining CD45 in Daudi cells. The cells were fixed with 80% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab200315 at 1/250 dilution (shown in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor® 647), at 1/250 dilution (shown in red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in Daudi cells fixed with 4% formaldehyde (10 min).

Equilibrium disassociation constant (KD)
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

Unpurified ab40763 showing negative staining in Skeletal muscle tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

Unpurified ab40763 showing positive staining in Normal colon lymphoid tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

Unpurified ab40763 showing positive staining in Normal spleen tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40763).

This IHC data was generated using the same anti-CD45 antibody clone, EP322Y, in a different buffer formulation (cat# ab40763).

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 counter-stained 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, and is shown in the inset.