Recombinant Anti-CD45 antibody [EPR20033] - BSA and Azide free (ab229292)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208022).

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208022).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208022).

Blocking/Dilution buffer: 5% NFDM/TBST.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208022).