Anti-TMEM106B antibody [EPR27525-70] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TMEM106B antibody [EPR27525-70] - BSA and Azide free (AB321971)

This data was developed using ab321970, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labelling TMEM106B with ab321970 at 1/500 (1.034 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in A549 cell line (shown in green) and no staining in TMEM106B KO A549 cell line. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-TMEM106B antibody [EPR27525-70] - BSA and Azide free (AB321971)

This data was developed using ab321970, the same antibody clone in a different buffer formulation.

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

This antibody cross-react with recombinant human TMEM106C by western blot.

All lanes:

Western blot - Anti-TMEM106B antibody [EPR27525-70] (<a href='/en-us/products/primary-antibodies/tmem106b-antibody-epr27525-70-ab321970'>ab321970</a>) at 1/1000 dilution

Lane 1:

His-tagged human TMEM106B recombinant fragment at 10 ng

Lane 2:

His-tagged human TMEM106C recombinant fragment at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 14 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-TMEM106B antibody [EPR27525-70] - BSA and Azide free (AB321971)

This data was developed using ab321970, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS.

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot : Anti-TMEM106B antibody (ab321970) staining at 1/1000 dilution, shown in green; Mouse anti-Tubulin antibody [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab321970 was shown to bind specifically to TMEM106B. Target of interest was observed at 40 kDa in wild-type A549 cell lysates (lane 1) with no signal observed at this size in TMEM106B knockout cell line (lane 2, knockout cell line ab273711 / knockout cell lysate ab273769). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

Negative control : Raji.

Exposure time : N/A

All lanes:

Western blot - Anti-TMEM106B antibody [EPR27525-70] (<a href='/en-us/products/primary-antibodies/tmem106b-antibody-epr27525-70-ab321970'>ab321970</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

TMEM106B knockout A549 whole cell lysate at 20 µg

Lane 3:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Raji (human burkitts lymphoma b lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 40 kDa

false