I've tried many different methods, but I have no signal. My current protocol (for any other protein it works very well, but not now) is a Bis-tris gel (8%), MOPS running buffer (running at 100v), in PAGE, and for transfer I use transfer buffer (tris, glycine, metOH), and PVDF (0.2) membrane (80v for 2h with ice pack).
There are no problems with my antibodies, I checked that with a dot blot. And when I prove a loading control, it works.
I stained membranes with ponceau and visualized the proteins, also when I stain with coomassie.
FMRP runs at 81kDa.
Can anybody help me?
Hi Erik, I have a few suggestions:
1. This could simply be a loading issue. Have you tried a range of loadings (total protein per lane) on the same gel to see if there is a threshold below which the signal will disappear? (This is quite common, and given you see a signal on your dot blot, I would definitely try this).
If there is no joy with that, then you could...
2. Try transfer at constant current, not constant voltage (I usually go for 250 mA for 60 min, or 400 mA for 30-45 min).
3. I don't know if FMRP is an appreciably hydrophobic protein, but if it is, try dropping MeOH content of transfer buffer from 20% to 10%.
4. Try PFA-fixing the membrane after transfer before blocking. This is a last resort, as it very rarely makes a difference, but in a few rare cases, it can.
Good luck for getting your protocol working!
Which antibody are you using? By far the best antibody is Abcam #17722. What tissue or cell type are you probing? Could be a loading issue if you're loading tissue of cells lysate with lo expression of FMRP.
I use a tris-glycine running buffer and transfer to PVDF for 1hr at 100V. You should see three bands around 80kD. The attached image is from 20ug hippocampal lysate loaded 1:10,000 primary 1:5,000 secondary. 30 second exposure. I use western lightning plus (http://www.perkinelmer.com/Catalog/Family/ID/Western%20Lightning%20Chemiluminescence%20Reagent%20Plus%20Enhanced%20Luminol).
Have you gotten it to work? Also might want to make sure it's not something simple like accidentally trying to save your secondary and adding sodium azide to it which will inactivate the HRP.
cb
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