Mouse anti flag m5

A discontinuous SDS-PAGE was performed using 7% acrylamide gels for high molecular weight proteins (NCAM, polysialylated NCAM) and 10% for low molecular weight proteins (polySTs, IBs). Samples were incubated for 5 min at 95 °C or for 10 min at 65 °C if polySia was supposed to be detected. In this case an additional incubation step with endosialidase [84 (link)] was inserted for the negative control. Protein blotting was performed via semi-dry blot on a PVDF membrane using 48 mM Tris, 39 mM Glycerin in H₂O. After blocking for 30 min with 5% skimmed milc detection of the PolySTs were performed with mouse anti-Flag M5 (Santa Cruz Biotechnology) and goat anti-mouse IgG Fc peroxidase labelled antibody (Jackson ImmunoResearch).
The transfected CHO cell lysates were directly analysed by immunoblotting with mouse anti-polySia 735 antibody [85 (link)] (polySia detection) and goat anti-mouse IgG Fc peroxidase labelled antibody (Jackson ImmunoResearch). NCAM was detected with rat anti-NCAM H28 antibody [86 (link)] and goat anti-rat peroxidase conjugated antibody (Santa Cruz Biotechnology).
Primary antibodies were incubated for 2 h and secondary antibodies for 1 h, diluted in 2% skimmed milc. Staining was performed with Sigma Fast 3,3’-Diaminobenzidin (DAB) Tetrahydrochlorid/Sigma Fast Metal Enhancer (Sigma Aldrich) according to supplier instructions.

Cells were fixed with 4% paraformaldehyde (10 min at room temperature) followed by permeabilization with 0.1% Triton X-100 for 10 min. Cells were washed three times with PBS/3%BSA and blocked for 20 min with the same solution. The endoplasmic reticulum was visualized using mouse anti-calnexin AF18 (Abcam) and goat anti-mouse Cy3 labeled antibody (Dianova), the Golgi apparatus was visualized using rabbit anti-α-Mannosidase II [83 (link)] and goat anti-rabbit Cy3 labeled antibody (Dianova). The IBs were detected with mouse anti-c-myc 9E10 (Santa Cruz Biotechnology) and goat anti-mouse Cy3 labeled antibody (Dianova) or directly with goat anti-c-myc FITC conjugated antibody (Novus Biologicals). Flag-HA-tagged polySTs were detected with mouse anti-FLAG M5 (Santa Cruz Biotechnology) and goat anti-mouse Cy3 or goat anti-mouse FITC labeled antibody. Primary antibodies were incubated for 2 h and secondary antibodies for 1 h (diluted in 3% BSA/PBS) at room temperature. In between, cells were washed 5 times with PBS-0.05%Tween. For analysis by laser scanning confocal microscopy, cells were coated with Vectashield Mounting Medium (Vector Laboratories).