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

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 +4ºC. 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.