I load 10 ug total protein/lane and use orbital shaker for incubation/washing the membranes. Playing with different dilution of primary and secondary antibody is not helping.
I would recommend seeing if your protein is blowing through the membrane during transfer. Try double-stacking the membrane and blotting both for actin. If you see a sort of inverse pattern in the "back" membrane, then your protein is just going through due to a non-homogeneous field gradient on the transfer.
If this is the case, then the solution might be to reduce your transfer time or voltage/amps, or to see if your transfer apparatus needs repair.
Dear Amrit
I concur with Jordan's suggestion that is could be a physical transfer issue if confined to lanes in the middle of a gel/Protran membrane
To add to Jordan's answer:
1. Make sure when you construct the transfer stack and place your gel in apposition to your NC/ Protran that your role over with a roller or plastic pipette to ensure complete and uniform contact between gel and membrane
2. Also, make sure you have pre wetted membrane in methanol followed by washing in transfer buffer before constructing the stack
3. Make sure your membrane/ Protran (especially if original style nitrocellulose) is not more than 2 years old: Particularly with NC, after such time the membrane can become highly brittle (and slightly off white) and this can lead to the sorts of issues depicted in your gel image
4. Make sure you perform transfer with fresh transfer buffer or at the very least fresh methanol added to your stock Tris solution: Avoid using old transfer buffer already containing methanol
5. Finally, to check that transfer has occurred properly, make sure your whole molecular ladder has disappeared from your gel and is visible on your membrane. Further, stain your membrane with Ponceau S for about 2-5 min (and wash with TBS tween to remove background) to verify that total protein lysate is as strong in the middle of the gel as periphery (Now wash with distilled water to completely remove the red dye before constructing stack: Water will remove stain from the transferred protein lysate whereas PBS/TBS TBS Tween will only remove background staining where protein has not sequestered
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