DIG Northern blot background problem - (Jun/15/2007 )
My DIG northern blot works at last in my hand after one month keeping on trying. But my present problem is my blot has high background even in the un-blot region. I am using the DIG northern system from ROCHE, their protocol said you can expose the film for 24-48 hours. But in my hand, it is impossbile, because after 30 mins later, the film will be very very dark.
My procedure is as following, after fixed by UV light, the film is put into tube and perform prohybridization at 60 for 30 min. Then hybridize with DIG-PCR probe for 16 hours, wash with 2xSSC/0.1%SDS for 2 times 5min each, then wash with 0.5xSSC/0.1% SDS for 2 times 15 min each at 50 degree. Washing with washing buffer , then incubate with blocking buffer for 30 min, incubate with antibody (1:10000) for 30 min. Washing with washing buffer for 15 min 2 times. Add CDP-Star, cover with plastic film, incubate for 5 min, and then exposure to film.
Are there any problem with my protocol? Need I use 0.1xSSC/0.1SDS in my secondary washing after hybridization? How can they expose for 24 hour? Thank you in advance.
hello....
well, the background could be due many factors.
1-when you pre-hybidize did you put a piece of clean membrane when incubating the probe with church buffer. This step is done before the 16hs hybridization and it is supposed to avoid unespecific reactions of the probe with the membrane.
2-I think I wash for longer time than you do after hybridization.
I send my protocol so you can compare.
* Wash buffer 1 (800 ml)
0.2x SSC (20x) 8ml
0.1% SDS (10%) 8 ml
*wash buffer 2 (200 ml)
0.3% tween 20
fill up 200 ml with maleic buffer.( 0.1 M maleic acid, 0.15 M NaCl, pH 7.5 (NaOh), autoclave)
Wash the sample membrane (30min x 2) at room temeperature with wash buffer 1.
Wash the membrane with wash buffer 2 (slow shaking) at room temperature.
In a plastic bag place the sample membrane with 5 ml of blocking buffer for an hour (slow motion)
At the same time with the previous point, in a 15 ml tube, mix 5 ml of blocking buffer with 1 microl of DIG (digoxigenin) and introduce a small piece of new membrane. (For non-specific reaction). (30 min to 1 hour)
Wash the membrane with wash buffer 2 (15 min x3)
Reveal with BCIP or cdp-star.
3- have less exposition time ..maybe 5 or 10 min.
Hope it can help.
good luck
Thank you very much. I will try that. Your ideas to clean the probe and antibody with membrane are really new to me. Thank you very much.
well, the background could be due many factors.
1-when you pre-hybidize did you put a piece of clean membrane when incubating the probe with church buffer. This step is done before the 16hs hybridization and it is supposed to avoid unespecific reactions of the probe with the membrane.
2-I think I wash for longer time than you do after hybridization.
I send my protocol so you can compare.
* Wash buffer 1 (800 ml)
0.2x SSC (20x) 8ml
0.1% SDS (10%) 8 ml
*wash buffer 2 (200 ml)
0.3% tween 20
fill up 200 ml with maleic buffer.( 0.1 M maleic acid, 0.15 M NaCl, pH 7.5 (NaOh), autoclave)
Wash the sample membrane (30min x 2) at room temeperature with wash buffer 1.
Wash the membrane with wash buffer 2 (slow shaking) at room temperature.
In a plastic bag place the sample membrane with 5 ml of blocking buffer for an hour (slow motion)
At the same time with the previous point, in a 15 ml tube, mix 5 ml of blocking buffer with 1 microl of DIG (digoxigenin) and introduce a small piece of new membrane. (For non-specific reaction). (30 min to 1 hour)
Wash the membrane with wash buffer 2 (15 min x3)
Reveal with BCIP or cdp-star.
3- have less exposition time ..maybe 5 or 10 min.
Hope it can help.
good luck
i think background always dependent on waching step after antibody or enzyme binding (streptavidine) in biotin labeling.
wach more than 15 min after antibody and enzyme linking.
two : you must try many exposure time to the X-ray film. from 10 second to five min
we expose for 30 second maximum and its work very well