Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Smad3 antibody (ab28379)

Ab28379, at a 1/100 dilution, staining Smad3 in formalin fixed paraffin embedded human breast carcinoma sections by Immunohistochemistry.

Western blot - Smad3 antibody (ab28379)

All lanes : Anti-Smad3 antibody - ChIP Grade (ab28379) at 1/3000 dilution

Lane 1 : HGL-5 cells: no additives
Lane 2 : HGL-5 cells: + activin A
Lane 3 : HGL-5 cells: + Cyclophilin, siRNA Control
Lane 4 : HGL-5 cells: + activin A + Cyclophilin
Lane 5 : HGL-5 cells: non targeting, negativ control for siRNA
Lane 6 : HGL-5 cells: plus activin A, negative control for siRNA
Lane 7 : HGL-5 cells: plus siRNA Smartpool
Lane 8 : HGL-5 cells: + activin A +siRNA Smartpool
Lane 9 : Jurkat cells

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/50000 dilution

Predicted band size : 48 kDa
Observed band size : 58 kDa (why is the actual band size different from the predicted?)

This image is courtesy of an Abreview submitted by Mrs Birte Jablonski

ChIP - Smad3 antibody - ChIP Grade (ab28379)

Chromatin was prepared from the nuclear cell lysate of a human myofibroblast cell line according to the X-ChIP protocol; cross-linking with formaldehyde for 10 minutes. ab28379 was diluted 1/16 (RIPA 0.1% SDS) and incubated with the sample for 16 hours at 4°C. ab28379 IP was used as the positive control and rabbit IgG pool IP was used as the negative control. Smad3 expression was enhanced by 2ng/ml TGFbeta 2. The immunoprecipitated DNA was quantified by real time PCR.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Smad3 antibody - ChIP Grade (ab28379)

ICC/IF image of ab28379 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 (ab28379, 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.

Immunocytochemistry/ Immunofluorescence - Smad3 antibody - ChIP Grade (ab28379)

ab28379 staining Smad3 in human Human TII Pneumocyte A549 cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton x100 before blocking with 3% BSA was done for 1 hour at RT. Samples were incubated with primary antibody (1/200: in 3% BSA in 1x PBST) for 24 hours at 4°C. An TRITC-conjugated goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.

This image is a courtesy of Aaron Gardner