Anti-RNF20 antibody (ab32629)

  Western blot - RNF20 antibody (ab32629)

All lanes : Anti-RNF20 antibody (ab32629) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate (ab7902)
Lane 5 : MCF-7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 6 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size : 114 kDa
Observed band size : 120 kDa (why is the actual band size different from the predicted?)


    

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - RNF20 antibody (ab32629)

Image courtesy of Human Protein Atlas

ab32629 staining RNF20 in human testis, showing a distinct and strong staining pattern in ductus seminiferus cells. Paraffin embedded human skin tissue was incubated with ab32629 (1/50 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab32629 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

  Immunocytochemistry/ Immunofluorescence - RNF20 antibody (ab32629)

ab32629 (1/500) staining RNF20 in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100/PBS and counterstained with DAPI to highlight the nucleus (red). Please refer to abreview for further experimental details.

This image is courtesy of an Abreview submitted by Dr Kirk McManus

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  Immunocytochemistry/ Immunofluorescence - RNF20 antibody (ab32629)

ICC/IF image of ab32629 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32629, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.

  Western blot - Anti-RNF20 antibody (ab32629)

All lanes : Anti-RNF20 antibody (ab32629) at 1/1000 dilution

Lane 1 : Whole cell lysate prepared from Ntera2 cells
Lane 2 : Whole cell lysate prepared from Ntera2 cells + siRNA against RNF20 for 48 hours

Lysates/proteins at 20 µg per lane.

Secondary
HRP conjugated goat polyclonal at 1/10000 dilution
developed using the ECL technique

Predicted band size : 114 kDa
Observed band size : 120 kDa (why is the actual band size different from the predicted?)


Exposure time : 20 seconds

Image courtesy of an anonymous Abreview.

See Abreview