Recombinant Anti-TMPRSS2 antibody [EPR3862] - BSA and Azide free (ab242384)

All lanes : Anti-TMPRSS2 antibody [EPR3862] (ab109131) at 1/2000 dilution

Lane 1 : Wild-type LNCaP cell lysate
Lane 2 : TMPRSS2 knockout LNCaP cell lysate
Lane 3 : Wild-type Caco-2 cell lysate
Lane 4 : TMPRSS2 knockout Caco-2 cell lysate
Lane 5 : PC-3 cell lysate
Lane 6 : DU 145 cell lysate
Lane 7 : Human Prostate cell lysate
Lane 8 : Human Colon cell lysate
Lane 9 : Human Lung cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 25, 55 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-TMPRSS2 antibody [EPR3862] staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109131 was shown to bind specifically to TMPRSS2. A band was observed at 55, 25 kDa in wild-type LNCaP and at 25 kDa in Caco-2 cell lysates with no signal observed at this size in TMPRSS2 knockout LNCaP cell line ab273745 (knockout LNCaP cell lysate ab275499) and TMPRSS2 knockout Caco-2 cell line ab273737 (knockout Caco-2 cell lysate ab277340). To generate this image, wild-type and TMPRSS2 knockout 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.

Anti-TMPRSS2 antibody [EPR3862] (ab109131) at 1/5000 dilution (Purified) + Human prostate lysate

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 54 kDa

54kDa represents full-length TMPRSS2 and 25kDa is the cleaved form. The molecular weights observed are consistent with what have been described in the literature (PMID: 11245484).

This data was developed using ab109131, the same antibody clone in a different buffer formulation.

ab109131 staining TMPRSS2 in Human prostate adenocarcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/1000). An undiluted HRP-conjugated mouse anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin. PBS was used in the negative control rather than the Primary antibody.

This data was developed using ab109131, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human prostatic adenocarcinoma using ab109131, unpurified, at a dilution of 1/500.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.