All lanes : Anti-GFP antibody [9F9.F9] (ab1218) at 1 mg/ml (1 hour at room temperature)

Lane 1 : HeLa lysate 50ng with BLOTTO, overnight at 4°C
Lane 2 : HeLa lysate 100ng with BLOTTO, overnight at 4°C
Lane 3 : HeLa lysate 500ng with BLOTTO, overnight at 4°C

Blocking peptides at 5 % per lane.

Secondary
All lanes : IRDye® 800 conjugated Goat anti-mouse IgG (H+L), 45 minutes at room temperature at 1/2500 dilution

Additional bands at: 27 kDa. We are unsure as to the identity of these extra bands.

 

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TIRFM responses to prolonged stimulation with 10 mM Ca²⁺

C. Cells expressing BS-wt were stimulated with 0.5 or 10 mM Ca²⁺ for 60 min, 37°C, followed by fixation, permeabilization, and staining with anti-GFP antibody (secondary antibody tagged with Alexa-568). Confocal images of SEP and anti-GFP fluorescence are shown along with the merged image. D. Experiment as in C for cells expressing BS-5A mutant.

HEK-293 cells were seeded on glass coverslips and transiently transfected with BS-wt or BS-5A cDNAs. Cells were incubated in DMEM containing 0.5 or 10 mM Ca²⁺ for 1 hr at 37°C, 5% CO₂. Following incubation, cells were immediately fixed in 4% paraformaldehyde for 10 min, RT, permeabilized with 0.2% Triton X-100 (10 min), blocked with 5% serum and 0.1% BSA then incubated with monoclonal anti-GFP antibody (Abcam), followed by goat anti-mouse secondary antibody conjugated to Alexa-568.

All lanes : Anti-GFP antibody [9F9.F9] (ab1218) at 1/1000 dilution (2 hours at 25°C)

Lane 1 : J774 with Milk, 2 hours at 25°C
Lane 2 : J774+pEGFP-N1 with Milk, 2 hours at 25°C

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

Secondary
All lanes : Goat anti-mouse HRP at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Additional bands at: 26 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute

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