Anti-MICA + MICB antibody [EPR22031]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling MICA with ab224702 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human lung cancer (PMID : 28334733) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue labeling MICA with ab224702 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human ovary cancer (PMID : 28334733) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling MICA with ab224702 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on epithelium of human stomach (PMID : 28334733) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• WB

Supplier Data

Western blot - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Bands between 50-60kDa represent the glycosylated form. After PNGase F treatment, the de-glycosylated form appears around 40kDa.

The molecular weight observed is consistent with what has been described in the literature (PMID : 10359807; PMID : 9396860).

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MICA + MICB antibody [EPR22031] (ab224702) at 1/1000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia cell line from bone marrow), whole cell lysate at 20 µg

Lane 2:

K562 whole cell lysate treated with PNGase F at 20 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate treated with PNGase F at 20 µg

Lane 5:

HUVEC (human umbilical vein endothelial cell line), whole cell lysate at 20 µg

Lane 6:

U266B1 (human multiple myeloma B lymphocyte cell line), whole cell lysate at 20 µg

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 1/50000 dilution

Predicted band size: 43 kDa

Observed band size: 40 kDa,50-60 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Western blot : Anti-MICA antibody [EPR22031] (ab224702) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab224702 was shown to bind specifically to MICA. A band was observed at 43 kDa in wild-type A549 cell lysates with no signal observed at this size in MICA knockout cell line. To generate this image, wild-type and MICA knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-MICA + MICB antibody [EPR22031] (ab224702) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MICA knockout A549 cell lysate at 20 µg

Lane 3:

U-2 OS cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Lane 5:

MICA protein <a href='/en-us/products/proteins-peptides/recombinant-human-mica-protein-active-ab182709'>ab182709</a> cell lysate at 0.2 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: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-MICA + MICB antibody [EPR22031] (AB224702)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MICA + MICB antibody [EPR22031] (ab224702) at 1/5000 dilution

Lane 1:

His-tagged MICB recombinant protein expressed in E.coli system (aa23-383 with 310-330 replaced by GGGS), 10 ng

Lane 2:

His-tagged MICA recombinant protein expressed in E.coli system (aa24-383 with 308-328 replaced by GGGGS) 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 1/100000 dilution

Predicted band size: 43 kDa

false

Exposure time: 15s