重组Anti-IL-11RA抗体[EPR5446]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-11RA antibody [EPR5446] (AB125015)

Overlay histogram showing K562 cells stained with unpurified ab125015 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab125015, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in K562 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL-11RA antibody [EPR5446] (AB125015)

Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling IL-11RA with Purified ab125015 at 1 : 100 dilution (8.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-11RA antibody [EPR5446] (AB125015)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling IL-11RA with purified ab125015 at 1 : 250 dilution (3.2 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) ab97051
was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-11RA antibody [EPR5446] (AB125015)

Unpurified ab125015 at 1/500 staining IL-11RA in paraffin embedded Human Kidney tissue by immunohistochemistry.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IL-11RA antibody [EPR5446] (AB125015)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling IL-11RA with Purified ab125015 at 1/80 dilution (10 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• I-ELISA

Lab

Indirect ELISA - Anti-IL-11RA antibody [EPR5446] (AB125015)

ELISA analysis of Human IL-11RA recombinant protein at 1000 ng/mL with ab125015. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-IL-11RA antibody [EPR5446] (AB125015)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of A673 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab125015 (1/2000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab125015 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-IL-11RA antibody [EPR5446] (ab125015) at 1/2000 dilution

All lanes:

A673 whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

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

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-IL-11RA antibody [EPR5446] (AB125015)

The expression pattern is consistent with what has been described in the literature (PMID : 26876177).

Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-IL-11RA antibody [EPR5446] (ab125015) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

WEHI-231 (mouse B cell lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 3:

M1 (mouse myeloid leukemia myeloblast) whole cell lysate at 20 µg

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

Mouse testis tissue lysate at 20 µg

Lane 6:

Mouse thymus tissue lysate at 20 µg

Lane 7:

Mouse lymph node tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

Observed band size: 42 kDa,45 kDa

false

Exposure time: 3min

• WB

Unknown

Western blot - Anti-IL-11RA antibody [EPR5446] (AB125015)

All lanes:

Western blot - Anti-IL-11RA antibody [EPR5446] (ab125015) at 1/1000 dilution

Lane 1:

293T lysate at 10 µg

Lane 2:

Saos2 lysate at 10 µg

Lane 3:

A673 lysate at 10 µg

Lane 4:

PC3 lysate at 10 µg

Lane 5:

Hut78 lysate at 10 µg

Lane 6:

K562 lysate at 10 µg

Lane 7:

TF1 lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-IL-11RA antibody [EPR5446] (AB125015)

All lanes:

Western blot - Anti-IL-11RA antibody [EPR5446] (ab125015) at 1/10000 dilution

Lane 1:

A673 (Human muscle Ewing's Sarcoma ) whole cell lysates at 20 µg

Lane 2:

PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

HuT-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysates at 20 µg

Lane 4:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-IL-11RA antibody [EPR5446] (AB125015)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.