Hi all,

Thank you in advance.

I labelled my membrane receptor (a GPCR 41 kDa; approx 80 kDa with SNAP at N-terminus and nLuc at C-terminus) with SNAP-AlexaFluor-488 (surface/non-permeable) and SNAP-647-SiR (permeable to the membrane). Lysed cells, collected total protein (stored on ice), stored at -20 dC for a week. Ran 10 uL supernatant on mPAGE™ 4-12% Bis-Tris Precast Gel, 10x8 cm. Electrophoresed first at 60V for 6 min (for protein to enter the gel) and then at 200 V for 33 min at room temperature in MOPS running buffer. Post-electrophoresis washed gel with tap water three times for 5 minutes. Scanned on Amersham Typhoon gel scanner using filter Cy2 (488 nm), Cy5 (635 nm), and Cy3 (532 nm). I see no problem with the Cy2 channel, but with the other two channels the images are weird - the gel appears granular, with white patches.

Note: while setting up the tank (just before loading the samples and filling the running buffer) I think I first slightly overtightened to create a seal but stopped and loosened it.

Please find the attached images

Please let me know if you need more information from my end.

Thank you once again.