All lanes : Anti-Syntrophin antibody [1351] (ab11425) at 5 µg/ml

Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lane 2 : Skeletal Muscle (Rat) Tissue Lysate
Lane 3 : Skeletal Muscle (Mouse) Tissue Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?
Additional bands at: 26 kDa, 42 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 12 minutes

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling Syntrophin (green) with ab11425 at 1/20. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Immunocytochemistry/Immunofluorescence analysis of A2058 cells labeling Syntrophin (green) with ab11425 at 1/20. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Immunocytochemistry/Immunofluorescence analysis of 293 cells labeling Syntrophin (green) with ab11425 at 1/20. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Overlay histogram showing SH-SY5Y cells stained with ab11425 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab11425, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Syntrophin was immunoprecipitated using 0.5mg Mouse Skeletal Muscle tissue lysate, 5µg of Mouse monoclonal to Syntrophin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Skeletal Muscle tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab11425.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 58kDa; Syntrophin