Anti-Syntrophin antibody [1351]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Syntrophin antibody [1351] (AB11425)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Syntrophin antibody [1351] (AB11425)

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.

• Flow Cyt

Unknown

Flow Cytometry - Anti-Syntrophin antibody [1351] (AB11425)

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 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Syntrophin antibody [1351] (AB11425)

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.

• IP

Unknown

Immunoprecipitation - Anti-Syntrophin antibody [1351] (AB11425)

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

All lanes:

Immunoprecipitation - Anti-Syntrophin antibody [1351] (ab11425)

Predicted band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-Syntrophin antibody [1351] (AB11425)

Protein samples were electrophoresed by SDS-PAGE using a 12% Bis-Tris gel. Resolved proteins were then transferred onto a nitrocellulose membrane.The membrane was probed with the relevant primary and secondary antibodies following blocking with 5% skimmed milk.

All lanes:

Western blot - Anti-Syntrophin antibody [1351] (ab11425) at 1 µg/mL

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 30 µg

Lane 2:

PC-3 (human prostate adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Mouse IgG (H+L) (HRP) at 1/4000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

true

• WB

Unknown

Western blot - Anti-Syntrophin antibody [1351] (AB11425)

All lanes:

Western blot - Anti-Syntrophin antibody [1351] (ab11425) at 5 µg/mL

Lane 1:

Human skeletal muscle tissue lysate - total protein (<a href='/en-us/products/unavailable/human-skeletal-muscle-tissue-lysate-total-protein-ab29330'>ab29330</a>) at 20 µg

Lane 2:

Skeletal Muscle (Rat) Tissue Lysate at 20 µg

Lane 3:

Skeletal Muscle (Mouse) Tissue Lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 54 kDa

Observed band size: 26 kDa,42 kDa,58 kDa

true

Exposure time: 12min

• WB

CiteAb

Western blot - Anti-Syntrophin antibody [1351] (AB11425)

Syntrophin western blot using anti-Syntrophin antibody [1351] ab11425. Publication image and figure legend from Kraner, S. D., Novak, K. R., et al., 2012, Skelet Muscle, PubMed 22935229.

ab11425 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab11425 please see the product overview.

NaV1.4 co-precipitates with members of the dystrophin associated protein complex (DAPC). Using an antibody generated against a peptide corresponding to the highly conserved III-IV linker region (pan NaV 1.x), sodium channels from NP40-solubilized control and CIM skeletal muscle membranes were immunoprecipitated. The immunoprecipitated (IP) control (Con) or CIM materials were resolved on SDS-PAGE gels and probed in western blots with the indicated antibodies. As a negative control, the IP was performed in the presence of blocking peptide (IP/pep), the same sequence used as the antigen; and as a positive control, starting membranes were used (Memb). (A) A full-panel western with the NaV 1.4-specific monoclonal antibody LD3 is shown on the left in comparison to a protein gel stained with SYPRO Ruby protein stain on the right. The denatured antibody heavy chain (Ab HC) and light chain (Ab LC) are present in the immunoprecipitated samples, as best seen in the SYPRO stained gel. A large number of proteins co-precipitate with the sodium channel (NaV 1.x), and many of these are shown to be specific since they are absent in the peptide control. (B) Using antibodies against plectin, dystrophin (dys), neuronal nitric oxide synthase (nNOS), syntrophin (syn), and β-dystroglycan (β-dys) confirms that many components of the DAPC are present in the control and CIM IPs. ( C) Quantification of the data from panel B. For each antibody in each CoIP, the signal for the control was set as 100% and the signal for the CIM was determined relative to this. The average of the signals for the CIM : control for each antibody is shown, and error bars are SEM ( n =6). For some antibodies, notably the sodium channel antibodies, approximately equal signals were seen in control and CIM CoIPs. For other antibodies, notably dystrophin and syntrophin, there were slightly elevated amounts of these proteins present in the CIM. Finally, for the nNOS and β-dystroglycan, there was considerably more protein in the CIM CoIPs. These results are summarized in cartoon form in ( D) which shows ‘tightly’ associated proteins in grey, and ‘loosely’ associated proteins in white. The protein associations shown in the cartoon are based on work carried out by a number of investigators, which previously demonstrated that NaV 1.x channels associate with proteins of the DAPC through their consensus S/TXV-COOH C-termini [26,27]. The dynamic regulation of the signaling protein nNOS in CIM suggests that it may play a role in the disease process, including affecting the inactivation gating of the adjacent sodium channels.

false