Western blotting -smearing effect - (Sep/11/2006 )
The last couple of times that I have performed western blotting, I get a smearing effect only in the region above my protein of interest. The entire area is black. Everything was done the same way I used to do and I have gotten good blots before. Could it be due to bad sample loading buffer???
Any advice is appreciated.
could you attach a picture? It would help
Thank you for the reply. I have attached a pic. Hope this helps.
I never saw something like that before.
Your protein of interest, is it the lower band, or the band in the middle just under the background?
Did you do a ponceau red staining of the membrane after transfer?
what is the quantity of protein you loaded?
I guess that the signal is strong, and comes very fast : you could dilute more your antibodies, but it would not solve completely your problem.
It could be a problem of denaturation? then you would have aggregates?
I have also a question; are you using discontinuous SDS-gels, and is the upper "black" phase the area belonging to the upper sampling gel phase? Is everything ok with the gels (composition, ionic strength, pH (= Cl-))? If not, entering the separating gel phase may fail. May use a continous gradient gel without sampling phase.
Thanks for your input.
I am also not sure why that happens. I figured that bad denaturing conditions might be the case. I made new sample buffer and store it at 4 degrees.
I am not sure what discontinous gel means.
discontinuous gel is when you pour first the separation gel which is at pH8.8 and then you pour some stacking gel wich is at pH 6.8 (on the well side). the role of stacking gel is to concentrate all your proteins in a thin band.
Usually I cut the stacking gel before I transfer. It is said that it can make background (?!) I never observed that when I didn't cut (I cut not to have troubles with the wells, making bubbles and so on)