Western blot with smeared bands - (Jul/24/2014 )

Hi everybody,

I got a problem in my western blots which is really annoying me.

I dont know why I always obtain smear bands. (Picture from attached file)

May anyone help me, please?

a few possibilities (may be others and/or multiple causes):

too much protein

incompletely solubilized protein

aggregated protein

overdeveloped blot

antibody too concentrated

Thanks you so much,

Attached files below is SDS PAGE. 

I think it's not too much protein loaded.

My protein is assembled protein.

Developed time: 1 min

Antibody concentration: 1st 1:5000 ; 2nd 1:7000

I don't know the smeared bands come from.

remember that immunostaining is a lot more sensitive than coomassie (especially coomassie g). you could reduce the concentration of primary antibody.

you can try spinning out insoluble proteins prior to addition of sds sample buffer.

you might want to look into adjusting the denaturing conditions for your sample.

do you boil at 95-100C? if so, then how long? over boiling can cause aggregation, even in the presence of sds and 2-mercaptoethanol (or dtt).

you may want to try 60-70C for ~20 minutes.