blurry, smeared bands on my western blots - new to the world of the westerns (Jun/18/2007 )
hey everyone,
This is my first post. I've been reading up on all these forums hoping to pick up some tricks that will help me with my westerns.
I've been running a few westerns and I can never get sharps bands. The folks in my lab have been telling me it's my samples, but I don't know what that means and how to fix it.
So here's what I've been doing. I'm running my samples in 8% Tris-glycine gels at 100V which takes about 2 hours for the dye front to reach the bottom. Then transfer for 1.5 hours at 0.25A onto a nitrocellulose membrane. Block in 5% non-fat milk in TBS-T (tween) at room temp for 1 hour. Incubate in primary Ab overnight at 4 degrees. Wash with TBS-T. Incubate in secondary for 1 hr. Detected with ECL detection reagents.
I've tried to upload a pic of my blot
http://lindaplong.myphotoalbum.com/view_ph...bum03&id=LP
I was hoping someone can give me some advice on what I can change to make this western run better!
Thanks!
How have you been preparing your samples? What are your samples and how much have you been loading?
I'm working with the sciatic nerve of rats. I take fresh sciatic nerves, chop it up as much as I can with a razor blade (the connective tissue is tricky. it always gets caught in the teeth of the polytron). homogenize it in solution A (50mM Tris, 4mM EDTA, ddH2O, protease inhibitor, pH 7.4) Centrifuge at 1000g for 10 mins, collect supernatant. Centrifuge again at 50 000g for 30 mins. Resuspend pellets in solution B (50mM Tris, 0.2mM EDTA, ddH2O, protease inhibitor, ph 7.4) by vortexing and sonicating.
Then do a protein determination. I've been loading 15uL of sample which turns out to be about 13ug (micrograms) of protein.
Looks to me like you just may not be using the correct percent gel and that's why you get a smear. What size is your protein? You also may be overloading your sample, maybe try a few different concentrations and run them in parallel.
So, I may be off base here, but your marker lane (that is what the third lane with bands is right?) looks really good, and I think that by and large this controls for issues with running the gel and transfers. I really don't think your bands look bad, especially on the right hand side of the gel, and your background is very nice which is also a good sign... Is this by chance a glycosylated or phosphorylated protein? You may expect to see a slightly diffuse band when you have modified proteins like those, so I say it looks good, maybe reduce the amount in lanes 1-2, especially lane 2 (the one just to the left of what i referred to as the marker lane) but the other lanes I think look alot like what you normally see with phosphorylated or glycosylated proteins... do you have some reason to expect crisp bands (another persons gel etc) because you will not or will rarely see nice crisp bands like you see in DNA gels when doing western blotting... Anyway I don't do straight westerns very often but that is my two cents...
HTH and good luck!