Anti-CD45 antibody [EP322Y] - BSA and Azide free

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

This data was developed using ab40763, the same antibody clone in a different buffer formulation.Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell, Left) / Jurkat (Human T cell leukemia T lymphocyte, Right) cells labelling CD45 with ab40763 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control : 293T. (PMID : 16005866)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab40763 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left).

PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interactions. Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min, followed by the addition of antibody ab40763 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/10,500)) for 30 min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

This data was developed using ab40763, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat cells labelling CD45 with ab40763 at 1/100 (6.1 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranouse staining in Jurkat cells and no staining in 293T cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-CD45 antibody [EP322Y] (<a href='/en-us/products/primary-antibodies/cd45-antibody-ep322y-ab40763'>ab40763</a>) at 1/5000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

PTPRC CRISPR-Cas9 edited Jurkat cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 147 kDa

Observed band size: 160-220 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

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).

• OI-RD Scanning

Supplier Data

OI-RD Scanning - Anti-CD45 antibody [EP322Y] - BSA and Azide free (AB214437)

Equilibrium disassociation constant (KD)
Learn more about KD

Go here to learn more about KD :

https : //www.abcam.com/index.html?pageconfig=resource&rid=15749

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