Anti-Syntaxin 6 antibody [3D10] (ab12370)

  Immunocytochemistry/ Immunofluorescence - Syntaxin 6 antibody [3D10] (ab12370)

ab12370 at 1/200 staining a mouse macrophage cell line by ICC/IF. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 24 hours. A FITC conjugated donkey anti-mouse antibody was used as the secondary.

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  Western blot - Syntaxin 6 antibody [3D10] (ab12370)

All lanes : Anti-Syntaxin 6 antibody [3D10] (ab12370) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Brain (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 30.6 kDa
Observed band size : 30.6 kDa
Additional bands at : 26 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 8 minutes

  Western blot - Syntaxin 6 antibody [3D10] (ab12370)

Predicted band size : 30.6 kDa


Intracellular membranes from CHO cells were separated on a 10–24% discontinuous Nycodenz gradient. Western Blotting was then performed using a range of antibodies including ab12370 at a 1/1000 dilution. An HRP-conjugated anti-mouse secondary was used at a 1/6000 dilution.

Image from Jonas MC et al, J Cell Sci. 2010 Oct 1;123(Pt 19):3378-88. Epub 2010 Sep 7, Fig 1, doi:10.1242/jcs.068841.

  Immunoprecipitation - Anti-Syntaxin 6 antibody [3D10] (ab12370)

Syntaxin 6 was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 10µg of Mouse monoclonal to Syntaxin 6 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 whole 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 ab12370.
Secondary: Protein G-HRP at 1/500 dilution.
Band: 25kDa:Syntaxin 6.

  Flow Cytometry - Anti-Syntaxin 6 antibody [3D10] (ab12370)

Overlay histogram showing PC12 cells stained with ab12370 (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 (ab12370, 2µg/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.