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recombinant human IL-6 produced in E.coli.
IL-6 synonyms: plasmacytoma growth factor (PCT-GF),interferon-a-2 (IFN-a2), monocyte derived human B cellgrowth factor, B cell stimulating factor (BSF-2),hepatocyte stimulating factor (HSF), and interleukinhybridoma/plasmacytoma-1 (IL-HP1).
Our Abpromise guarantee covers the use of ab6672 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/500 - 1/2000. Can be blocked with IL6 peptide (ab133194).|
|ELISA||1/1000 - 1/5000.|
|IHC-P||1/400 - 1/800.|
|RIA||1/4000 - 1/8000.|
|IP||1/400 - 1/800.|
|IHC-Fr||1/400 - 1/800.|
|ICC/IF||1/500. See Abreview.|
Tissue lysates were denatured for 10-15 minutes at 90ºC. ab6672 was incubated overnight at 4ºC and the secondary antibody for 1 hour at RT.
ab6672 detects a band at 25 kDa in Human lung tissue lysate and Mouse spleen tissue lysate, however the signal in mouse tissue is significantly lower. It also binds strongly to a protein at ~55 kDa in Human lung tissue extracts, which we believe represents a glycosylated form of IL6. ab6672 also detects several bands in Human lung tissue lysate within the region of 30-40 kDa. These may represent heteromers of IL6.
This image is courtesy of an Abreview submitted by Francesco Elia Marino
IHC image of IL6 staining in human lung formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6672, 1/400, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ICC/IF image of ab6672 stained THP1 cells. The cells were treated using phorbol 12-myristate 13-acetate (PMA) to induce differentiation and encourage cell adhesion to plate, Lipopolysaccharide (LPS) (100ng/ml for 6 hours) to increase expression of IL6, and Befreldin (300ng/ml for 3 hours) to inhibit secretion of IL6. Following treatment the cells were 100% Methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6672 at 1in500 dilution overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti-rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
ab6672 staining IL6 (red) in Mouse mesenchimal SC cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.5% Tween-20 and blocked with 3% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/500 in 3% BSA) for 1 hour. An Alexa Fluor® 555-conjugated Donkey anti-rabbit IgG polyclonal (1:500) was used as the secondary antibody. Blue (DAPI) - nuclei.