All lanes : Anti-Sec8 antibody [14G1] (ab13254) at 5 µg/ml
Lane 1 : Human brain tissue lysate - total protein (ab29466) Lane 2 : Human kidney tissue lysate - total protein (ab30203) Lane 3 : Human placenta tissue lysate - total protein (ab29745) Lane 4 : Brain (Mouse) Tissue Lysate
Lysates/proteins at 20 µg per lane.
Secondary 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 : 117 kDa Observed band size : 117 kDa Additional bands at : 45 kDa,52 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 8 minutes
Flow Cytometry-Anti-Sec8 antibody [14G1](ab13254)
Overlay histogram showing HeLa cells stained with ab13254 (red line). The cells were fixed with 80% methanol (5 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 (ab13254, 1µg/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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Immunocytochemistry/ Immunofluorescence - Anti-Sec8 antibody [14G1] (ab13254)Image from Barkefors I et al., J Biol Chem. 2011 Jul 8;286(27):24189-99. Epub 2011 May 12. Fig 4.; doi: 10.1074/jbc.M110.212209; July 8, 2011 The Journal of Biological Chemistry, 286, 24189-24199.
Immunofluorescence analysis of HMVECs, staining Sec8 with ab13254.
Cells were fixed in paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with blocking buffer. Samples were incubated with primary antibody (1/50) overnight at 4°C. An AlexFluor®-conjugated anti-mouse IgG (1/500) was used as a secondary antibody.
Sec8 was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Mouse monoclonal to Sec8 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 Brain 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab13254. Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution. Band: 117kDa, non specific bands - 45kDa: We are unsure as to the identity of this extra band; Sec8
Winkelmann A et al. Changes in neural network homeostasis trigger neuropsychiatric symptoms. J Clin Invest124:696-711 (2014).
Read more (PubMed: 24430185) »
Barkefors I et al. Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells. J Biol Chem286:24189-99 (2011).
Read more (PubMed: 21566143) »