I am currently comparing snail-1 expression in esophageal cancer cell lines. My protein load was 30-40 ug, the primary snail-1 antibody (cell signaling) was 1:1000, and the secondary anti-rabbit antibody was 1:5000. The snail-1 band was quite faded and needed 1 hour exposure time.
How can I improve my western blot results?
try 1:500 dilution. also overnight incubation with primary antibody helps. u can also use ECL+ solution, which gives strong bands.
good luck
you try to use primary antibody 1:200-1:500 and incubate overnight in 4c with shaker. If the band is not clear, you can use secondary con. 1:3000 cople with primary antibody that i sugggest.
You can try to load more protein sample. Also, I find that if I use a narrow toothed comb when pouring my gel (15 well vs 10 well) one can enhance signal for low abundance targets.
Snail-1 is tightly regulated and quickly degraded by GSK3 phosphorylation. If you add LiCl you can prevent this degredation.
i would put the membrane for overnight and try to increase the exposure time of flim, and if you have Fuji machine will so helpful. good luck
Hi Kee,
Which cell signaling Ab are you using? I would follow their protocol exactly for your particular antibody. For 2 of the snail Abs they have listed you are supposed to block in 5% milk in TBS/Tween and dilute the primary in 5% BSA-TBS/Tween followed by O/N incubation at 4 degrees. The other one is to be diluted in 5% milk. I have found if you follow their protocol I usually get very good results. You may also have a problem with protein stability as stated above. Hope that helps! Good luck.
Hi Kee,
Do you have any idea of the amount of Snail-1 you are loading? Usually there is far less of the protein of your interest compared to others. I have at times loaded 75ug total protein per lane. So, increasing the amount might be worth a shot.
If you have enough sample, run multiple lanes, then separate them and incubate each with different concentration of antibody, from 1:50 to the recommenced 1:1000 and see how it goes. It will solve a lot of confusion usually.
With the LiCl, I will do a comparison to see whether it is needed or not.
Blocking with a lower concentration of milk, 2% instead of 5% o/n in 4C, might also help, if you are sure the blocking solution should be milk. I have seen it is sometimes worth a try.
As Tina said, also double check the buffer you have to dilute your antibody in.
Longer incubation with the primary antibody, up to 72hrs in 4C, might also be worth trying.
Good Luck
Thanks for all recommendations..
for your information, i use 1:2000 dilution, cell signaling Snail-1 rabbit antibody..
my milk % is 5% in PBST
I incubated overnight. I will try longer incubation.
When you use milk, it is the casein in it that blocks the areas of the nitrocellulose paper that no protein is attached to, so that your antibody, as a protein, does not attach to those empty areas giving you a false signal. In some cases people believe you can over-block your blot, hence the casein will shadow your protein of interest. Hence reducing the concentration and slowing the attachment/reaction by putting in lower temperature might solve the problem.
Hi:
I've been running snail from Novus Biologicals (Cat# NBP1-19529) @ 1:4K v/v. I'm loading 10ug. My secondary is from Cell Signaling @ 1:10K. Make sure you leave the primary on for a minimum of 16 hours, and your secondary for 1.25 hours. Good luck!!
The above blot I used 1:1K, but since, I ran a dilution series and was able to take it out to 1:4K. I block with 5% milk.
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