Recombinant Anti-CD45 antibody [EPR20033] (ab208022)

False colour image of Western blot: Anti-CD45 antibody [EPR20033] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab208022 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue sections labeling CD45 with purified ab208022 at 1/5000 dilution (0.14 µg/ml). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.

Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Heat mediated antigen retrieval was performed with Tris/EDTA pH9 buffer (ab93684).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue sections labeling CD45 with purified ab208022 at 1/5000 dilution (0.14 µg/ml). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.

Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Heat mediated antigen retrieval was performed with Tris/EDTA pH9 buffer (ab93684).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 30 seconds; Lane 2: 5 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

293T and MCF7 are reported to be CD45 negative cell lines (PMID: 22978632& 24602188).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 15 seconds.

CD45 was immunoprecipitated from 0.35 mg of J774A.1 (Mouse reticulum cell sarcoma macrophage cell line) whole cell lysate with ab208022 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab208022 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: J774A.1 whole cell lysate 10µg (Input).

Lane 2: ab208022 IP in J774A.1 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208022 in J774A.1 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.