Anti-TMEM175 抗体 [EPR24415-47] (ab300457)

All lanes : Anti-TMEM175 antibody [EPR24415-47] (ab300457) at 1/1000 dilution

Lane 1 : Wild-type HeLa (boiled) cell lysate
Lane 2 : TMEM175 knockout HeLa (boiled) cell lysate
Lane 3 : U-87 MG (boiled) cell lysate
Lane 4 : Wild-type HeLa (unboiled) cell lysate
Lane 5 : TMEM175 knockout HeLa (unboiled) cell lysate
Lane 6 : U-87 MG (unboiled) cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 40-50 kDa why is the actual band size different from the predicted?

Anti-TMEM175 antibody [EPR24415-47] (ab300457) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab300457 was shown to bind specifically to TMEM175 only when the lysate samples were not boiled. A band was observed at 40-50 kDa in wild-type HeLa cell lysates with no signal observed at this size in TMEM175 knockout cell line ab265571 (knockout cell lysate ab258722). To generate this image, wild-type and TMEM175 knockout HeLa 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Immunohistochemical analysis of paraffin-embedded human brain tissue labelling TMEM175 with ab300457 at 1/500 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human brain. The section was incubated with ab300457 for 30 mins at room temperature. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human endometrial cancer tissue labelling TMEM175 with ab300457 at 1/500 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human endometrial cancer. The section was incubated with ab300457 for 30 mins at room temperature. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.