Anti-TMEM175 antibody [EPR24415-47] (BSA and Azide free)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TMEM175 antibody [EPR24415-47] (BSA and Azide free) (AB300458)

This data was developed using ab300457, the same antibody clone in a different buffer formulation.

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).

• WB

Lab

Western blot - Anti-TMEM175 antibody [EPR24415-47] (BSA and Azide free) (AB300458)

This data was developed using ab300457, the same antibody clone in a different buffer formulation. 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.

All lanes:

Western blot - Anti-TMEM175 antibody [EPR24415-47] (<a href='/en-us/products/primary-antibodies/tmem175-antibody-epr24415-47-ab300457'>ab300457</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (boiled) cell lysate at 20 µg

Lane 2:

TMEM175 knockout HeLa (boiled) cell lysate at 20 µg

Lane 3:

U-87 MG (boiled) cell lysate at 20 µg

Lane 4:

Wild-type HeLa (unboiled) cell lysate at 20 µg

Lane 5:

TMEM175 knockout HeLa (unboiled) cell lysate at 20 µg

Lane 6:

U-87 MG (unboiled) cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 6:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 56 kDa

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