ab14106 stained Hela cells. The cells were 100% methanol fixed for 5 minutes, permeabilized in 0.1% PBS-Triton X-100 for 5 min and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14106 at 1µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed used at a 2/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour 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.
Immunocytochemistry/ Immunofluorescence - Anti-SM22 alpha antibody (ab14106)This image is courtesy of an anonymous Abreview.
ab14106 staining SM22 alpha in mouse muscle cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 10% serum for 30 minutes at 25°C. Samples were incubated with primary antibody (1/100 in PBS) for 18 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/1000.
All lanes : Anti-SM22 alpha antibody (ab14106) at 2 µg/ml
Lane 1 : HeLa Whole Cell Lysate Lane 2 : HeLa Nuclear Extract Lane 3 : Human Skeletal Muscle Tissue Lysate
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution
Predicted band size: 23 kDa Observed band size: 23 kDa
Lane 1 - 3 : SM22 alpha antibody (ab14106) at 1 ug/ml
Lane 1 : HeLa Whole Cell Lysate at 20 ug Lane 2 : HeLa Nuclear Extract at 20 ug Lane 3 : Human Muscle lysate at 20 ug
Secondary Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution
Observed band size : 23kD
The band was completed abolished by peptide blocking with ab16067 (immunising peptide) - not shown.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SM22 alpha antibody (ab14106)This image is courtesy of an anonymous Abreview.
ab14106, at 1/100, staining SM22 alpha in chicken smooth muscle tissue sections by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Sections were methacarn (60% MeOH, 30% choloform, 10% acetic acid) fixed, prior to blocking in 2% serum for 5 minutes at 25°C and then incubated with ab14106, for 1 hour at 25°C. Alexa fluor® 488 goat polyclonal to rabbit Ig, diluted 1/100, was used as the secondary antibody.
All lanes : Anti-SM22 alpha antibody (ab14106) at 1 µg/ml
Lane 1 : Human colon tissue lysate - total protein (ab30051) Lane 2 : Colon (Mouse) Tissue Lysate Lane 3 : Colon (Rat) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 23 kDa Observed band size: 23 kDa Additional bands at: 115 kDa, 20 kDa, 36 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 5 seconds
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab14106 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.