Western Blot Troubles - (May/29/2007 )
hi all,
I often observed the following phenomenon (see attachment) after western blotting (amidoblack staining). Could anyone say me why my blots look like this. I suggest that this is no kind of air-inclusion. But maybe some of you also have or had experience with this kind of trouble.
thank you all very much
I often observed the following phenomenon (see attachment) after western blotting (amidoblack staining). Could anyone say me why my blots look like this. I suggest that this is no kind of air-inclusion. But maybe some of you also have or had experience with this kind of trouble.
thank you all very much
I have never seen such a Wb; do you always use the same blotting apparatus? may it is out of order; or in other words: I think of an irregular electric field...
do you presoak your membrane? are you sure that you soak the entire membrane? you may have a bubble under the membrane during the soak so the pores aren't properly filled where the bubble is. then when you put the membrane on the gel you may not have a bubble there but there is still poor electrical contact with the gel.
Continue Mdfenko suggestions use a little amount of methanol in water for good and fast membrane presoak
You have one air bubble in the top of lane 7. Round and clear.
The low MW smearing is due to poor physical or electrical contact between gel and blot.
The low MW smearing is due to poor physical or electrical contact between gel and blot.
Could this contact probem due to incomplete pretreatment with methanol?
The low MW smearing is due to poor physical or electrical contact between gel and blot.
Could this contact probem due to incomplete pretreatment with methanol?
yes
you have to put ur memebrane for 10 seconds in methanole then wash 1x5 with water then at least 10 minutes in transfer buffer conating methanol
The low MW smearing is due to poor physical or electrical contact between gel and blot.
Could this contact probem due to incomplete pretreatment with methanol?
yes
you have to put ur memebrane for 10 seconds in methanole then wash 1x5 with water then at least 10 minutes in transfer buffer conating methanol
thanks,
only for 10sec? I always did it like that. The problem is: I always pre-treat my membrane according to the recommendations of the manufacturer (amersham, hybond-p). And therefore it is hard to understand why this problem occurs from time to time.
is it possible to presoak it for a longer time? does this make sense?
well to tell you the truth o presoak it in methanol for 1or 2 minutes then wash once 5 times and then soak it in transfer buffer containing 20%methanol for as long as it take , sometimes i leave it for one hour.
Are you using a semi-dry blotter or a wet blot? Thats usually seen in semi-dry blots when the electrode plates are not entirely in contact or the papers are not soaked enough...
D