Anti-MICB antibody [EPR26362-65]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MICB antibody [EPR26362-65] (AB300485)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling MICB with ab300485 at 1/50 (9.62ug/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 membranous staining in K-562 cell lineNegative control : Daudi (PMID : 19404979 ) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-MICB antibody [EPR26362-65] (AB300485)

Flow cytometric analysis of 293T (human embryonic kidney epithelial cell, Left) / Daudi (human Burkitt's lymphoma lymphoblast, Middle) / K562 (human chronic myelogenous leukemia lymphoblast, Right) cells labelling MICB with ab300485 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.Negative control : 293T, Daudi (PMID : 28154561).

• IP

Supplier Data

Immunoprecipitation - Anti-MICB antibody [EPR26362-65] (AB300485)

MICB was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate 10 ug with ab300485 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300485 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate 10 ug Lane 2 : ab300485 IP in K562 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab300485 in K562 whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

Lane 1:

Immunoprecipitation - Anti-MICB antibody [EPR26362-65] (ab300485) at 1/30 dilution

Lane 2:

Immunoprecipitation - Anti-MICB antibody [EPR26362-65] (ab300485) at 1/1000 dilution

All lanes:

K562 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-MICB antibody [EPR26362-65] (AB300485)

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

This antibody does not cross-react with human MICA.

All lanes:

Western blot - Anti-MICB antibody [EPR26362-65] (ab300485) at 1/1000 dilution

Lane 1:

Purified his-tagged human MICB recombinant protein at 10 ng

Lane 2:

Purified his-tagged human MICA recombinant protein at 10 EU/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

Predicted band size: 43 kDa

Observed band size: 37-50 kDa

false

Exposure time: 7.75s

• WB

Supplier Data

Western blot - Anti-MICB antibody [EPR26362-65] (AB300485)

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

MICB is a glycoprotein of approximately 61 kDa and detected as two 37-42 kDa bands after treated with Protein Deglycosylation MIX II.

The bands beneath the target band (37 kDa) are reported as deglycosylated cleaved soluble MICB.

Molecular weight observed is consistent with described in the literature (PMID : 19147769).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-MICB antibody [EPR26362-65] (ab300485) at 1/1000 dilution

Lane 1:

Untreated K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 15 µg

Lane 2:

K562 whole cell lysate treated with Protein Deglycosylation Mix II at 15 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 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 37-61 kDa

true

Exposure time: 103s

• WB

Supplier Data

Western blot - Anti-MICB antibody [EPR26362-65] (AB300485)

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

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

Negative control : Daudi, 293T (PMID : 28154561).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-MICB antibody [EPR26362-65] (ab300485) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 40 µg

Lane 2:

U266B1 (human multiple myeloma B lymphocyte) whole cell lysate at 40 µg

Lane 3:

Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate at 40 µg

Lane 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 40 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>)

Predicted band size: 43 kDa

Observed band size: 37-61 kDa

true

Exposure time: 48s