Trouble Western Blotting a 360kDa Protein

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yosoytueres
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Trouble Western Blotting a 360kDa Protein

  Hello,

I have been trying for weeks to western blot a 360kDa membrane protein. I've followed suggestions from online posts but I haven't been successful.

I have prepared 3 different lysates (Thermo Scientific M-per and Mem-Per kits and one using RIPA buffer) using three different cell lines (Two of which should have this protein of interest).

I am currently using a 4-15% gradient gel, I load about 60 ug of my samples, run the gel at 150V for 2.5 hrs, transfer onto nitrocellulose membrane over night in tris-glycine based buffer with 0.05% SDS and 20% methanol overnight at 30V and then for 1 more hr at 70V. (I've also tried other transfer times and voltages as well as transfer buffer with only 0.037% SDS)

Staining of my membrane after transfer with Ponceau stain does not show bands at the expected location but I have also stained my gel once after transfer and no bands are present at the expected location either.

Can anyone please offer any suggestions?? I am at a dead end. 

g a
g a's picture
 Hi Yosoytureres

 Hi Yosoytureres

If you are using semidry transfer method, then switch to wet transfer method.

Since you are unable to see the band in yr gel after transfer as well as on membrane........ Use a positive control  lane which you stain immediately after electrophoresis and rest you can use for Transfer.

Use an initial higher voltage followed with lower voltage for transfer.....  Although unlikely, but 360 kDa protein can also diffuse. Check for any intermitent air bubbles b/w membrane and gel.

Finally have you proceeded with detection??? Ponceau has poor senstivity where as regular ECL detection kit may detect yr protein on the membrane as it's sensitivity can go as low as atto molar levels.

All the Best