All lanes : Anti-IL-6R antibody [EPR24322-143] (ab271042) at 1/1000 dilution

Lane 1 : U266B1 (human multiple myeloma B lymphocyte) whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 52 kDa
Observed band size: 70-80 kDa why is the actual band size different from the predicted?

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

The observed MW is consistent with what has been described in the literature (PMID: 28276471, 21282511).

Negative control: K-562 (PMID: 8300137)

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 37 seconds

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U266B1 cells labelling IL-6R with ab271042 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in U266B1 cells. Negative control: K-562 (PMID: 8300137) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human thyroid carcinoma tissue labelling IL-6R with ab271042 at 1/2000 dilution (0.255 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cyoplasmics staining on human thyroid carcinoma. The section was incubated with ab271042 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling IL-6R with ab271042 at 1/2000 dilution (0.255 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cyoplasmics staining on human tonsil. The section was incubated with ab271042 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labelling IL-6R with ab271042 at 1/2000 dilution (0.255 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cyoplasmics staining on human placenta (PMID: 25658637). The section was incubated with ab271042 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

IL-6R was immunoprecipitated from 0.35 mg U266B1 (human multiple myeloma B lymphocyte) whole cell lysate 10 ug with ab271042 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271042 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: U266B1 (human multiple myeloma B lymphocyte) whole cell lysate 10 ug

Lane 2: ab271042 IP in U266B1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271042 in U266B1 whole cell lysate

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

Exposure time: 5.5 seconds