Anti-CD45 抗体 [EP322Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] (AB40763)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (AB40763)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (AB40763)

Unpurified ab40763 showing positive staining in normal spleen tissue.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD45 antibody [EP322Y] (AB40763)

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)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] (AB40763)

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (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 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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD45 antibody [EP322Y] (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.

• WB

Lab

Western blot - Anti-CD45 antibody [EP322Y] (AB40763)

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.

All lanes:

Western blot - Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution

All lanes:

Jurkat (Human T cell lymphoblast-like cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 147 kDa

Observed band size: 200 kDa

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Exposure time: 8min

• WB

Lab

Western blot - Anti-CD45 antibody [EP322Y] (AB40763)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution

All lanes:

Raji (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 147 kDa

Observed band size: 200 kDa

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• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-CD45 antibody [EP322Y] (ab40763) 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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• WB

Lab

Western blot - 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 knockout cell line ab274932 (knockout cell lysate ab274990). The band observed in the knockout 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 knockout 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] (ab40763) at 1/5000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

PTPRC knockout Jurkat cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

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• WB

Lab

Western blot - Anti-CD45 antibody [EP322Y] (AB40763)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD45 antibody [EP322Y] (ab40763) at 1/5000 dilution

All lanes:

HuT-78 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 147 kDa

Observed band size: 200 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (AB40763)

Unpurified ab40763 showing negative staining in skeletal muscle tissue.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD45 antibody [EP322Y] (AB40763)

Unpurified ab40763 showing negative staining in normal kidney tissue.

• OI-RD Scanning

Supplier Data

OI-RD Scanning - Anti-CD45 antibody [EP322Y] (AB40763)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.